System for discovering and producing polypeptides that cause TNF receptor shedding

ABSTRACT

The present invention relates to methods of regulating TNF activity indirectly by regulating the activity or concentration of TNF receptor releasing enzyme (TRRE). Preferably, the TRRE activity is regulated local to the site of the condition to be treated. In the case of diseases associated with elevated levels of TNF, such as rheumatoid arthritis, TRRE is administered to the site of inflammation in an amount sufficient to decrease the local levels of TNF. In the case of diseases, such as cancer, that benefit from increased levels of TNF, the level of TRRE is decreased at the disease site.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/964,747,filed Nov. 5, 1997, now U.S. Pat. No. 6,569,664 which claims benefit ofprovisional application Ser. No. 60/030,761, filed Nov. 6, 1996.

FIELD OF THE INVENTION

This invention relates to the purification and characterization of tumornecrosis factor (TNF) receptor (TNF-R) releasing enzyme (TRRE),compositions derived from the enzyme, and methods of use thereof.Modulation of TRRE levels indirectly modulates effective levels of TNF.The invention further relates to methods of treatment of pathologicalconditions caused or exacerbated by altered levels or activity of TNFsuch as inflammatory conditions including autoimmune diseases,infections, septic shock, obesity, cachexia, and conditions that areassociated with decreased levels or activity of TNF such as cancer.

BACKGROUND OF THE INVENTION

Tumor necrosis factor (TNF or TNF-α) and lymphotoxin (LT or TNF-β) arerelated cytokines that share 40 percent amino acid (AA) sequencehomology. Old (1987) Nature 330:602-603. These cytokines are releasedmainly by macrophages, monocytes and natural killer (NK) cells inresponse to broad immune reactions. Gorton and Galli (1990) Nature346:274-276; and Dubravec et al. (1990) Proc. Natl. Acad. Sci. USA87:6758-6761. Although initially discovered as agents inducinghemorrhagic necrosis of tumors, these cytokines have been shown to haveessential roles in both the inductive and effector phases of immunereactions and inflammation. The two cytokines cause a broad spectrum ofeffects on cells in vitro and tissues in vivo, including: (i) vascularthrombosis and tumor necrosis; (ii) inflammation; (iii) activation ofmacrophages and neutrophils; (iv) leukocytosis; (v) apoptosis; and (vi)shock. Beretz et al. (1990) Biorheology 27:455-460; Driscoll (1994) Exp.Lung Res. 20:473-490; Ferrante (1992) Immunol. Ser. 57:417-436; Golsteinet al. (1991) Immunol. Rev. 121:29-65; and van der Poll and Lowry (1995)Shock 3:1-12. For a review of the mechanism of action of TNF, seeMassague (1996) Cell 85:947-950. TNF has been associated with a varietyof disease states including various forms of cancer, arthritis,psoriasis, endotoxic shock, sepsis, autoimmune diseases, infections,obesity, and cachexia. Attempts have been made to alter the course of adisease by treating the patient with TNF inhibitors with varying degreesof success. For example, oxpentifylline did not alter the course ofCrohn's disease, a chronic inflammatory bowel disease. Bauditz et al.(1997) Gut 40:470-4. However, the TNF inhibitor dexanabinol providedprotection against TNF following traumatic brain injury. Shohami et al.(1997) J. Neuroimmun. 72:169-77.

Human TNF and LT mediate their biological activities, both on cells andtissues, by binding specifically to two distinct, although related,glycoprotein plasma membrane receptors of 55 kDa and 75 kDa (p55 and p75TNF-R, respectively). Holtmann and Wallach (1987) J. Immunol.139:151-153. The two receptors share 28 percent AA sequence homology intheir extracellular domains, which are composed of four repeatingcysteine-rich regions. Tartaglia and Goeddel (1992) Immunol. Today13:151-153. However, the receptors lack significant AA sequence homologyin their intracellular domains. Dembic et al. (1990) Cytokine 2:231-237.Due to this dissimilarity, they may transduce different signals and, inturn, exercise diverse functions.

Recent studies have shown that most of the known cellular TNF responses,including cytotoxicity and induction of several genes, may be attributedto p55 TNF-R activation. Engelmann et al. (1990) J. Biol. Chem.265:1531-1536; Shalaby et al. (1990) J. Exp. Med. 172:1517-1520; andTartaglia et al. (1991) Proc. Natl. Acad. Sci. USA 88:9292-9296. Inaddition, the p55 receptor controls early acute graft-versus-hostdisease. Speiser et al. (1997) J. Immun. 158:5185-90. In contrast,information regarding the biological activities of p75 TNF-R is limited.This receptor shares some activities with p55 TNF-R and specificallyparticipates in regulating proliferation of and secretion of cytokinesby T cells. Shalaby et al. (1990); and Gehr et al. (1992) J. Immunol.149:911-917. Both belong to an ever-increasing family of membranereceptors including low-affinity nerve growth factor receptor (LNGF-R),FAS antigen, CD27, CD30 (Ki-1), CD40 (gp50) and OX 40. Cosman (1994)Stem Cells (Dayt.) 12:440-455; Meakin and Shooter (1992) TrendsNeurosci. 15:323-331; Grell et al. (1994) Euro. J. Immunol.24:2563-2566; Moller et al. (1994) Int. J. Cancer 57:371-377; Hintzen etal. (1994) J. Immunol. 152:1762-1773; Smith et al. (1993) Cell73:1349-1360; Corcoran et al. (1994) Eur. J. Biochem. 223:831-840; andBaum et al. (1994) EMBO J. 13:3992-4001.

All of these receptors share a repetitive pattern of cysteine-richdomains in their extracellular regions. In accord with the pleiotropicactivities of TNF and LT, most human cells express low levels (2,000 to10,000 receptors/cell) of both TNF-Rs simultaneously. Brockhaus et al.(1990) Proc. Natl. Acad. Sci USA 87:3127-3131. Expression of TNF-R onboth lymphoid and non-lymphoid cells may be up and down-regulated bymany different agents, such as bacterial lipopolysaccharide (LPS),phorbol myristate acetate (PMA; a protein kinase C activator),interleukin-1 (IL-1), interferon-gamma (IFN-γ) and IL-2. Gatanaga et al.(1991) Cell Immunol. 138:1-10; Yui et al. (1994) Placenta 15:819-835;and Dett et al. (1991) J. Immunol. 146:1522-1526. Although expressed indifferent proportions, each receptor binds TNF and LT with equally highaffinity. Brockhaus et al. (1990); and Loetscher et al. (1990) J. Biol.Chem. 265:20131-20138. Initial studies showed that the complexes ofhuman TNF and TNF-R are formed on the cell membrane, internalizedwholly, and then either degraded or recycled. Armitage (1994) Curr.Opin. Immunol. 6:407-413; and Fiers (1991) FEBS Lett. 285:199-212.

TNF binding proteins (TNF-BP) were originally identified in the serumand urine of febrile patients, individuals with renal failure, cancerpatients, and even certain healthy individuals. Seckinger et al. (1988)J. Exp. Med. 167:1511-1516; Engelmann et al. (1989) J. Biol. Chem.264:11974-11980; Seckinger et al. (1989) J. Biol. Chem. 264:11966-11973;Peetre et al. (1988) Eur. J. Haematol. 41:414-419; Olsson et al. (1989)Eur. J. Haematol. 42:270-275; Gatanaga et al. (1990a) Lymphokine Res.9:225-229; and Gatanaga et al. (1990b) Proc. Natl. Acad. Sci USA87:8781-8784. In fact, human brain and ovarian tumors produced highserum levels of TNF-BP. Gatanaga et al. (1990a); and Gatanaga et al.(1990b). These molecules were subsequently purified, characterized, andcloned by different laboratories. Gatanaga et al. (1990b); Olsson et al.(1989); Schall et al. (1990) Cell 61:361-370; Nophar et al. (1990) EMBOJ. 9:3269-3278; Himmler et al. (1990) DNA Cell Biol. 9:705-715;Loetscher et al. (1990) Cell 61:351-359; and Smith et al. (1990) Science248:1019-1023. These proteins have been suggested for use in treatingendotoxic shock. Mohler et al. (1993) J. Immunol. 151:1548-1561; Poratet al. (1995) Crit. Care Med. 23:1080-1089; Fisher et al. (1996) N.Engl. J. Med. 334:1697-1702; Fenner (1995) Z. Rheumatol. 54:158-164; andJin et al. (1994) J. Infect. Dis. 170:1323-1326.

Human TNF-BP consist of 30 kDa and 40 kDa proteins found to be identicalto the N-terminal extracellular domains of p55 and p75 TNF-R,respectively. The 30 kDa and 40 kDa TNF-BP are thus also termed solublep55 and p75 TNF-R, respectively. Studies of these proteins have beenfacilitated by the availability of human recombinant 30 kDa and 40 kDaTNF-BP and antibodies which specifically recognize each form and allowquantitation by immunoassay. Heller et al. (1990) Proc. Natl. Acad. Sci.USA 87:6151-6155; U.S. Pat. No. 5,395,760; EP 418,014; and Grosen et al.(1993) Gynecol. Oncol. 50:68-77. X-ray structural studies havedemonstrated that a TNF trimer binds with three soluble TNF-R (sTNF-R)molecules and the complex can no longer interact with TNF-R. Banner etal. (1993) Cell 73:431-445. The binding of the trimer and sTNF-R,however, is reversible and these reactants are not altered as a resultof complex formation. At high molar ratios of sTNF-R to TNF, bothrecombinant and native human sTNF-R are potent inhibitors of TNF/LTbiological activity in vitro as well as in vivo. Gatanaga et al.(1990b); Ashkenazi et al. (1991) Proc. Natl. Acad. Sci. USA88:10535-10539; Lesslaur et al. (1991) Eur. J. Immunol. 21:2883-2886;Olsson et al. (1992) Eur. J. Haematol. 48:1-9; and Kohno et al. (1990)Proc. Natl. Acad Sci. USA 87:8331-8335.

Increased levels of TNF-R are also associated with clinical sepsis(septic peritonitis), HIV-1 infection, and other inflammatoryconditions. Kalinkovich et al. (1995) J. Interferon and Cyto. Res.15:749-757; Calvano et al. (1996) Arch. Surg. 131:434-437; and Ertel etal. (1994) Arch. Surg. 129:1330-1337. Sepsis, and septic shock affectthousands of patients every year and there is essentially no cure. Thislethal syndrome is caused primarily by lipopolysaccharides (LPS) ofGram-negative bacteria and superantigens of Gram-positive bacteria.Clinical symptoms are initiated primarily by the release of endogenousmediators, such as TNF, from activated lymphoid cells into thebloodstream. TNF induces production of a cascade of other cytokines,including IL-1, gamma-Interferon, IL-8, and IL-6. These cytokines, alongwith other factors, promote the clinical symptoms of shock. Recombinanthuman sTNF-R is currently being tested in clinical trials to blockTNF/LT activity in patients with septic shock and other conditions inwhich TNF and LT are thought to be pathogenic. Van Zee et al. (1992)Proc. Natl. Acad. Sci. USA 89:4845-4849. Balb/c mice, the primary animalmodel, and multiple techniques have been used to test the effects of TNFmodulators and other treatments on septic peritonitis. Jin et al. (1994)J. Infect. Dis. 170:1323-1326; Mohler et al. (1993) J. Immunol.151:1548-1561; Porat et al. (1995) Crit. Care Med. 23:1080-1089; andEchtenacher et al. (1996) Nature 381:75-77. Lipopolysaccharide-inducedshock has been shown to be ameliorated by FR167653, a dual inhibitor ofIL-1 and TNF production. Yamamoto et al. (1997) Eur. J. Pharmacol.327:169-174.

Attempts have been made to ameliorate the untoward effects of TNF bytreatment with monoclonal antibodies to TNF or with other proteins thatbind TNF, such as modified TNF receptors. Patients with sepsis or septicshock were treated with anti-TNF antibodies. Salat et al. (1997) Shock6:233-7. Some improvement in the clinical and histopathologic signs ofCrohn's disease were afforded by treatment with anti-TNF antibodies.Neurath et al. (1997) Eur. J. Immun. 27:1743-50; van Deventer et al.(1997) Pharm. World Sci. 19:55-9; van Hogezand et al. (1997) Scand. J.Gastro. 223:105-7; and Stack et al. (1997) Lancet 349:521-4. In thetreatment of experimental autoimmune encephalitis (EAE), an animal modelof the human disease multiple sclerosis (MS), treatment with TNF-Rfusion protein prevents the disease and the accompanying demyelination,suggesting the possible use of this treatment in MS patients. Klinkertet al. (1997) J. Neuroimmun. 72:163-8. Neither coagulation nor thefibrinolytic system was affected by an anti-TNF antibody in a study ofpatients with sepsis or septic shock. Satal et al. (1996) Shock 6:233-7.

Regulation of TNF expression is being tested in treatment of endotoxicshock. Mohler et al. (1994) Nature 370:218-220. Modulation of TNF-Ractivity is also being approached by the use of peptides that bindintracellularly to the receptor or other component in the process toprevent receptor shedding. PCT patent publications: WO 95/31544, WO95/33051; and WO 96/01642. Modulation of TNF-R activity is alsopostulated to be possible by binding of peptides to the TNF-R andinterfering with signal transduction induced by TNF. European PatentApplication EP 568 925.

While low levels of sTNF-R have been identified in the sera of normalindividuals, high levels have been found in the sera of patients withchronic inflammation, infection, renal failure and various forms ofcancer. Aderka et al. (1992) Lymphokine Cytokine Res. 11:157-159; Olssonet al. (1993) Eur. Cytokine Netw. 4:169-180; Diez-Ruiz et al. (1995)Eur. J. Haematol. 54:1-8; van Deuren (1994) Eur. J. Clin. Microbiol.Infect. Dis. 13 Suppl. 1:S12-6; Lambert et al. (1994) Nephrol. Dial.Transplant. 9:1791-1796; Halwachs et al. (1994) Clin. Investig.72:473-476; Gatanaga et al. (1990a); and Gatanaga et al. (1990b). Serumlevels of sTNF-R rise within minutes and remain high for 7 to 8 hoursafter the intravenous injection of human recombinant TNF or IL-2 intohuman cancer patients. Aderka et al. (1991) Cancer Res. 51:5602-5607;and Miles et al. (1992) Br. J. Cancer 66:1195-1199. Contrarily, serumsTNF-R levels are chronically elevated in cancer patients and may remainat high levels for years. Grosen et al. (1993). It is clear that sTNF-Rare natural inhibitors of these cytokines and regulate their biologicalactivity post secretion. Fusion proteins consisting of a sTNF-R linkedto a portion of the human IgG1 have also been developed for treatingrheumatoid arthritis and septic shock. Moreland et al. (1997) N. Eng. J.Med. 337:141-7; Abraham et al. (1997) JAMA 277:1531-8.

New evidence has yielded information on cellular regulation of secretedcytokines. The evidence indicates that cells release molecules whichresemble or contain the binding site of the specific membrane receptors.Massague and Pandiella (1993) Annul. Rev. Biochem. 62:515-541; and RoseJohn and Heinrich (1994) Biochem. J. 300:281-290. These soluble formsspecifically bind and, in the appropriate molar ratios, inactivate thecytokine by steric inhibition. Therefore, this may be a generalphenomenon responsible for the regulation of cytokines and membraneantigens.

Notably, in addition to TNF-R, various types of membrane molecules haveboth soluble and membrane forms, including (i) cytokine receptors, e.g.,IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, granulocyte-colonystimulating factor-R (G-CSF-R), granulocyte-macrophage-colonystimulating factor-R (GM-CSF-R), transforming growth factor-β-R(TGFβ-R), platelet-derived growth factor-R (PDGF-R), and epidermalgrowth factor-R (EGF-R); (ii) growth factors, e.g., TNF-(pro-TNF-α),TGF-α, and CSF-1; (iii) adhesion molecules, e.g., intracellular adhesionmolecule-1 (ICAM-1/CD54) and vascular cell membrane adhesion molecule(VCAM-1/CD106); (iv) TNF-R/NGF-R superfamily, e.g., LNGF-R, CD27, CD30,and CD40; and (v) other membrane proteins, e.g. transferrin receptor,CD14 (receptor for LPS and LPS binding protein), CD16 (FcγRIII), andCD23 (low-affinity receptor for IgE). Colotta et al. (1993) Science261:472-475; Baran et al. (1988) J. Immunol. 141:539-546; Mosley et al.(1989) Cell 59:335-348; Takaki et al. (1990) EMBO J. 9:4367-4374; Novicket al. (1989) J. Exp. Med. 170:1409-1414; Goodwin et al. (1990) Cell60:941-951; Renauld et al. (1992) Proc. Natl. Acad. Sci. USA89:5690-5694; Fukunaga et al. (1990) Proc. Natl. Acad. Sci. USA87:8702-8706; Raines et al. (1991) Proc. Natl. Acad. Sci. USA88:8203-8207; Lopez-Casillas et al. (1991) Cell 67:785-795; Tiesman andHart (1993) J. Biol. Chem. 268:9621-9628; Khire et al. (1990) Febs.Lett. 272:69-72; Kriegler et al. (1988) Cell 53:45-53; Pandiella andMassague (1991) Proc. Natl. Acad. Sci. USA 88:1726-1730; Stein et al.(1991) Oncogene 6:601-605; Seth et al. (1991) Lancet 338:83-84; Hahne etal. (1994) Eur. J. Immunol. 24:421-428; Zupan et al. (1989) J. Biol.Chem. 264:11714-11720; Loenen et al. (1992) Eur. J. Immunol. 22:447-455;Latza et al. (1995) Am. J. Pathol. 146:463-471; Chitambar (1991) Blood78:2444-2450; Landmann et al. (1992) J. Leukoc. Biol. 52:323-330;Huizinga et al. (1988) Nature 333:667-669; and Alderson et al. (1992) J.Immunol. 149:1252-1257.

In vitro studies with various types of cells have revealed that thereare two mechanisms involved in the production of soluble receptors andcell surface antigens. One involves translation from alternativelyspliced mRNAs lacking transmembrane and cytoplasmic regions, which isresponsible for the production of soluble IL4R, IL-5R, IL-7R, IL-9R,G-CSF-R, and GM-CSF-R. Rose-John and Heinrich (1994); and Colotta et al.(1993). The other mechanism involves proteolytic cleavage of the intactmembrane receptors and antigens, known as shedding. Proteolysis appearsto be involved in the production of soluble LNGF-R, TNF-R, CD27, CD30,IL-1R, IL-6R, TGFβ-R, PDGF-R, and CD14 (Id.).

Matrix metalloproteinases (MMPs) are a family of enzymes that includesinterstitial collagenase (MMP-1), 72 kDa and 92 kDa gelatinases (MMP-2and MMP-9), stromelysins 1, 2 and 3, neutrophil collagenase,metalloelastase, matrilysin, and gelatinase A. These enzymes aresecreted by cells within tissues and by infiltrating inflammatory cells.Collectively, they are capable of degrading most of the proteins in theextracellular matrix (ECM).

MMPs display different substrate specificities yet have severalproperties in common. They are all zinc-containing enzymes that requirecalcium for activity. They are secreted as zymogens and activated insitu, usually by release of an inhibitory N-terminal pro-piececontaining a single cysteine residue. The attached pro-piece is believedto coordinate with the zinc in the active site of the proteinase,thereby suppressing the proteolytic activity. Activation may beaccompanied by additional proteolytic cleavages that can generate activeenzymes of lower molecular weights. All members of the MMP family have ashort conserved region consisting of the HEXGH motif that provides twoZn-coordinating histidine residues and a glutamic acid residue that isconsidered part of the catalytic site. With few exceptions, MMPs alsocontain a hemopexin/vitronectin domain. The function of the hemopexindomain is unknown. For review see Ray and Stetter-Stevenson (1994) Eur.Respir. J. 7:2062-2072.

A variety of studies have indicated that MMPs are involved in tumorinvasion and metastasis. A number of methods have been utilized toassess the presence of MMPs in human tumor tissues and serum from cancerpatients. Positive correlations have been found between MMP expressionand tumor invasion and metastasis in vitro, as well as in in vivo animalmodels. Matrisian et al. (1991) Am. J. Med. Sci. 302:157-162; Sato etal. (1992) Oncogene 7:77-83; Lyons et al. (1991) Biochemistry30:1449-1456; Levy et al. (1991) Cancer Res. 51:439-444; Bonfil et al.(1989) J. Natl. Cancer Inst. 81:587-594; Sreenath et al. (1992) CancerRes. 52:4942-4947; and Powell et al. (1993) Cancer Res. 53:415-422. MMPshave been associated with the malignant phenotype in a wide variety ofhuman tissues, including lung, prostate, stomach, colon, breast, ovariesand thyroid, as well as squamous carcinoma of the head and neck.Matrisian et al. (1991); Sato et al. (1992); Levy et al. (1991); andLyons et al. (1991). To date, the proposed role of MMPs in cancer hasbeen limited to tissue remodeling in invasion and metastasis.

The MMPs are inhibited by members of the family of tissue inhibitors ofmetalloproteinases (TIMPs, e.g., TIMP-1, TIMP-2, and TIMP-3), which bindat the active site and block access to substrate. Matrix remodeling,which occurs during various normal and pathological processes depends ona critical balance between activated MMPs and inhibiting TIMPs. Forreviews of MMPs and their inhibitors see Alexander and Werb (1991), In:Cell Biology of Extracellular Matrix, ed. Hay, Plenum Press, New York,pp. 205-302; Murphy et al. (1991) Br. J. Rheumatol. 30:25-31; Woessner(1991) FASEB J. 5:2145-2154; Matrisian (1992) Bioessays 14:455-463;Birkedal-Hansen et al. (1993) Crit. Rev. Oral Biol. and Med. 4:197-250;and Denhardt et al. (1993) J. Pharmacol. Ther. 59:329-341.

Recent studies suggest that metalloproteases may be involved in thecleavage of both TNF-Rs, LNGF-R, IL-6R, pro-TNF-α, VCAM-1, and CD30 andare thereby responsible for the production of the soluble forms. Croweet al. (1995) J. Exp. Med. 181:1205-1210; Mullberg et al. (1995) J.Immunol. 155:5198-5205; Bjomberg et al. (1995) Scand. J. Immunol.42:418-424; DiStefano et al. (1993) J. Neurosci. 13:2405-2414; Mohler etal. (1994) Nature 370:218-220; Gearing et al. (1994) Nature 370:555-557;McGeehan et al. (1994) Nature 370:558-561; Leca et al. (1995) J.Immunol. 154:1069-1077; and Hansen et al. (1995) Int. J. Cancer (1995)63:750-756. Interestingly, a MMP is suggested to be responsible for thecleavage of pro-TNF-α. Gearing et al. (1994); and Gearing et al. (1995)J. Leukoc. Biol. 57:774-777. In addition, levels of serum matrixmetalloproteinase 1 and 3 in rheumatoid arthritis patients were reducedfollowing anti-TNF antibody therapy. Brennan et al. (1997) Br. J.Rheumatology 36:643-50. Anti-TNF antibodies have also been used tosuppress fever, inflammation and the acute-phase response in juvenilechronic arthritis and rheumatoid arthritis cases, and to reverseendotoxin shock in rats. Elliott et al. (1997) Br. J. Rheumatology36:589-93; Maini et al. (1997) Apmis 105:257-63; and Boillot et al.(1997) Crit. Care Medicine 25:504-11. One MMP inhibitor, GM-6001,prevents the release of TNF both in vitro and in vivo. Solorzano et al.(1997) Shock 7:427-31.

A number of MMP inhibitors have been described and the use thereof hasalso been suggested for treating various pathologic indications,including: angiogenesis; wound healing; gum disease; skin disorders;keratoconus; inflammatory conditions; rheumatoid arthritis; cancer;corneal and skin ulcers; cardiovascular disease; central nervous systemdisorders; and diabetes. U.S. Pat. Nos. 5,268,384 and 5,270,326; PCTpublications WO 94/22309, WO 95/09913, WO 90/11287, WO 90/14363; EPpatents 211 077, 623 676; and Naito et al. (1994) Int. J. Cancer58:730-735; Watson et al. (1995) Cancer Res. 55:3629-3633; Davies et al.(1993) Cancer Res. 53:2087-2091; Brown (1995) Advan. Enzyme Regul.35:293-301; Sledge et al. (1995) J. Natl. Cancer Inst. 87:1546-1550;Conway et al (1995) J. Exp. Med. 182:449-457; and Docherty et al. (1992)TibTech 10:200-207. However, the ability to treat arthritis byinhibiting matrix metalloproteases has been questioned. Vincenti et al.(1994) Arth. & Rheum. 37:1115-1126.

Both soluble p55 and p75 TNF-R do not appear to be generated fromprocessed mRNA, since only full length receptor mRNA has been detectedin human cells in vitro. Gatanaga et al. (1991). Carboxyl-terminalsequencing of the human soluble p55 TNF-R indicates that a cleavage sitemay exist between Asn 172 and Val 173. Gullberg et al. (1992) Eur. J.Cell. Biol. 58:307-312. This evidence is supported by the finding thathuman TNF-R with the mutation at Asn 172 and Val 173 was not released aseffectively as native TNF-R on COS-1 cells transduced with cDNA of humanTNF-R. Gullberg et al. (1992). The cytoplasmic portion of TNF-R does notappear to play an important role in releasing the soluble receptor formsfrom transduced COS-1 cells. COS-1 cells release sTNF-R even whentransduced with cDNA of human p55 TNF-R which expresses only theextracellular domain but not the cytoplasmic domain. (Id.) sTNF-Rshedding is not affected by dexamethasone, gold sodium thiomalate, orprostaglandin E2. Seitz et al. (1997) J. Rheumatology 24:1471-6.Collectively, these data support the concept that human sTNF-R areproduced by proteolytic cleavage of membrane TNF-R protein.

It would be useful to purify and characterize the protease that cleavesTNF-R and results in the generation of sTNF-R. The purification andcharacterization of this proteinase will reveal the role of sTNF-R inhost-tumor interactions and in treatment of pathogenic conditionsmediated or exacerbated by TNF. Although claims have been made to theTRRE (EP 657 536), analysis of the claimed protein sequences given byBLAST Protein Sequence Homology Search reveals that they match theTNF-R. This may be due to the use of a TNF-R affinity column duringprotein purification. Thus, the nature of the protein and its DNA and AAsequences have not yet been elucidated.

In spite of numerous advances in medical research, cancer remains thesecond leading cause of death in the United States. In theindustrialized nations, roughly one in five persons will die of cancer.Traditional modes of clinical care, such as surgical resection,radiotherapy and chemotherapy, have significant failure rates,especially for solid tumors. Failure occurs either because the initialtumor is unresponsive, or because of recurrence due to regrowth at theoriginal site and/or metastases. Even in cancers such as breast cancerwhere the mortality rate has decreased, successful intervention relieson early detection of the cancerous cells. The etiology, diagnosis andablation of cancer remain a central focus for medical research anddevelopment.

Neoplasia resulting in benign tumors can usually be completely cured byremoving the mass surgically. If a tumor becomes malignant, asmanifested by invasion of surrounding tissue, it becomes much moredifficult to eradicate. Once a malignant tumor metastasizes, it is muchless likely to be eradicated.

The three major cancers, in terms of morbidity and mortality, are colon,breast and lung. New surgical procedures offer an increased survivalrate for colon cancer. Improved screening methods increase the detectionof breast cancer, allowing earlier, less aggressive therapy. Lung cancerremains largely refractory to treatment.

Excluding basal cell carcinoma, there are over one million new cases ofcancer per year in the United States alone, and cancer accounts for overone half million deaths per year in this country. In the world as awhole, the five most common cancers are those of lung, stomach, breast,colon/rectum, and uterine cervix, and the total number of new cases peryear is over 6 million. Only about half the number of people who developcancer die of it.

Melanoma is one of the human diseases for which there is an acute needof new therapeutic modalities. It is a particularly aggressive form ofskin cancer, and occurs in increased frequency in individuals withregular unguarded sun exposure. In the early disease phases, melanoma ischaracterized by proliferation at the dermal-epidermal junction, whichsoon invades adjacent tissue and metastasizes widely. Once it hasmetastasized, it is often impossible to extirpate and is consequentlyfatal. Worldwide, 70,000 patients are diagnosed annually with melanomaand it is responsible for 25,000 reported deaths each year. The AmericanCancer Society projects that by the year 2000, 1 out of every 75Americans will be diagnosed with melanoma.

Neuroblastoma is a highly malignant tumor occurring during infancy andearly childhood. Except for Wilm's tumor, it is the most commonretroperitoneal tumor in children. This tumor metastasizes early, withwidespread involvement of lymph nodes, liver, bone, lung, and marrow.While the primary tumor is resolvable by resection, the recurrence rateis high.

Small cell lung cancer is the most malignant and fastest growing form oflung cancer and accounts for 20-25% of new cases of lung cancer.Approximately 60,000 cases are diagnosed in the U.S. every year. Theprimary tumor is generally responsive to chemotherapy, but is followedby wide-spread metastasis. The median survival time at diagnosis isapproximately 1 year, with a 5 year survival rate of 5-10%.

Breast cancer is one of the most common cancers and is the third leadingcause of death from cancers in the United States with an annualincidence of about 182,000 new cases and nearly 50,000 deaths. In theindustrial nations, approximately one in eight women can expect todevelop breast cancer. The mortality rate for breast cancer has remainedunchanged since 1930. It has increased an average of 0.2% per year, butdecreased in women under 65 years of age by an average of 0.3% per year.Preliminary data suggest that breast cancer mortality is beginning todecrease, probably as a result of increased diagnoses of localizedcancer and carcinoma in situ. See e.g., Marchant (1994) ContemporaryManagement of Breast Disease II: Breast Cancer, In: Obstetrics andGynecology Clinics of North America 21:555-560; and Colditz (1993)Cancer Suppl. 71:1480-1489.

Non-Hodgkin's B cell lymphomas are cancers of the immune system that areexpected to afflict approximately 225,000 patients in the United Statesin 1996. These cancers are diverse with respect to prognosis andtreatment, and are generally classified into one of three grades. Themedian survival of the lowest grade is 6.6 years and the higher gradecancers have much lower life expectancy. Virtually all non-Hodgkin's Bcell lymphomas are incurable. New diagnoses of non-Hodgkins lymphomashave increased approximately 7% annually over the past decade, with52,700 new diagnoses estimated for 1996. The increase is due in part tothe increasing prevalence of lymphomas in the AIDS patient population.

In spite of the difficulties, effective cures using anticancer drugs(alone or in combination with other treatments) have been devised forsome formerly highly lethal cancers. Most notable among these areHodgkin's lymphoma, testicular cancer, choriocarcinoma, and someleukemias and other cancers of childhood. For several of the more commoncancers, early diagnosis, appropriate surgery or local radiotherapyenables a large proportion of patients to recover.

Current methods of cancer treatment are relatively non-selective.Surgery removes the diseased tissue, radiotherapy shrinks solid tumorsand chemotherapy kills rapidly dividing cells. Chemotherapy, inparticular, results in numerous side effects, in some cases severeenough to preclude the use of potentially effective drugs. Moreover,cancers often develop resistance to chemotherapeutic drugs.

Recently, a method of in situ treatment of cancers, particularlypancreas, has been shown to be efficacious. The method involves creatingan mixed lymphocyte reaction (MLR) between the host (cancer patient's)peripheral blood lymphocytes and a donor's allogeneic lymphocytes andadministering the MLR directly to the tumor. This method is describedmore fully, for example, in WO 93/20186 and JP 62096426. In the case oflarge solid tumors, administration of the MLR is preceded by resectionof the tumor.

Like cancers, weight problems are also associated with TNF. TNF islinked to the three factors contributing to body weight control: intake,expenditure, and storage of energy. Administration of either TNF orIL-1, for example, induces a decrease in food intake. Rothwell (1993)Int. J. Obesity 17:S98-S101; Arbos et al. (1992) Mol. Cell. Biochem.112:53-59; Fargeas et al. (1993) Gastroenterology 104:377-383;Plata-Salaman et al. (1994) Am. J. Physiol. 266:R1711-1715; Schwartz etal. (1995) Am. J. Physiol. 269:R949-957; and Oliff et al. (1987) Cell50:555-563. Interestingly, TNF may have a key roles in both extremes ofweight problems. Abnormalities in its activity may lead to obesity;changes in its production result in the opposite effect, cachexia.Argilés et al. (1997) FASEB J. 11:743-751.

Cachexia is pathological weight loss generally associated with anorexia,weakness, anemia, asthenia, and loss of body lipid stores and skeletalmuscle protein. This state often accompanies burns, trauma, infection,and neoplastic diseases. Lawson et al. (1982) Annu. Rev. Nutr.2:277-301; Argilés et al. (1988) Mol. Cell. Biochem. 81:3-17; andOgiwara et al. (1994) J. Surg. Oncol. 57:129-133. TNF concentrations areelevated in many patients with cachexia. Scuderi et al. (1986) Lancet2:1364-65; Grau et al. (1987) Science 237:1210-1212; and Waage et al.(1986) Scand. J. Immunol. 24:739-743. TNF inhibits collagen alpha I geneexpression and wound healing in a murine model of cachexia. Buck et al.(1996) Am. J. Pathol. 149:195-204. In septicemia (the invasion ofbacteria into the bloodstream), increased endotoxin concentrations mayraise TNF levels, causing cachexia. Beutler et al. (1985) Science229:869-871; Tracey et al. (1987) Nature 330:662-664; and Michie et al.(1988) New Engl. J. Med. 318:1481-1486. During cachexia, the loss ofwhite adipose tissue is caused by the decreased activity of lipoproteinlipase (LPL); TNF lowers the activity of this enzyme. Price et al.(1986) Arch. Biochem. Biophys. 251:738-746; Cornelius et al. (1988)Biochem. J. 249:765-769; Fried et al. (1989) J. Lipid. Res.30:1917-1923; Semb et al. (1987) J. Biol. Chem. 262:8390-8394; and Evanset al. (1988) Biochem. J. 256:1055-1058. Fat tissue loss is alsoassociated with an increase in lipase activity and inhibition of glucosetransport; TNF is also linked to both of these changes. Kawakami et al.(1987) J. Biochem. 331-338; Feingold et al. (1992) Endocrinology130:10-16; and Hauner et al. (1995) Diabetologia 38:764-771. TNFmediates hypertriglyceridaemia associated with cachexia. Dessi et al.(1995) Br. J. Cancer 72:1138-43. TNF also participates in the proteinwasting, loss of skeletal muscle and loss of nitrogen associated withcachexia. Costelli et al. (1993) J. Clin. Invest. 92:2783-2789; Floreset al. (1989) J. Clin. Invest. 83:1614-1622; Goodman (1991) Am. J.Physiol. 260:E727-730; Zamir et al. (1992) Arch. Surg. 127:170-174;Llovera et al. (1993) J. Natl. Cancer Inst. USA 85:1334-1339; andGarcia-Martinez et al. (1993) FEBS Lett. 323:211-214.

TNF has additional, related roles. It is involved in thermogenesis,particularly nonshivering thermogenesis in brown adipose tissue (BAT),which has an elevated level in cachexia. Nicholls (1983) Biosci. Rep.3:431-441; Rothwell (1993) Int. J. Obesity 17:S98-S101; Bianchi et al.(1989) Horm. Metab. Res. 21:11; and Oudart et al. (1995) Can. J.Physiol. Pharmacol. 73:1625-1631. TNF has also been implicated innon-insulin-dependent (type II) diabetes. Hotamisligil et al. (1995) J.Clin. Invest. 95:2409-2415; Arner (1996) Diabetes Metab. 13:S85-S86;Spiegelman et al. (1993) Cell 73:625-627; Saghizadeh et al. (1996) J.Clin. Invest. 97:1111-16; and Hofmann et al. (1994) Endocrinology134:264-270. These data help explain how TNF mediates the oppositeeffects of obesity and cachexia. TNF has functional similarities tothose of leptin, which has been proposed to be an “adipostat.” Zhang etal. (1994) Nature 372:425432; Phillips et al. (1996) Nature Genet.13:18-19; and Madej et al. (1995) FEBS Lett. 373:13-18. Like leptin, forexample, TNF is expressed and secreted by adipocytes and can travel tothe brain. TNF administration also results in an increase in circulatingleptin concentrations. Grunfeld et al. (1996) J. Clin. Invest.97:2152-57. It is possible to reconcile the participation of TNF inobesity and cachexia. TNF can be considered one of many signals comingfrom adipose tissue that participate in the feedback mechanism thatinforms the hypothalamic center about the state of the adipocyte energydepot. It probably counteracts excessive energy intake and is able tostimulate thermogenesis either directly or by increasing sympatheticactivity. TNF released by adipose tissue will also stimulate lipolysis,decrease LPL activity, decrease the expression of the glucosetransporter GLUT4, and inhibit lipogenesis in the adipocyte, thuscontributing to the maintenance (but not increased fat deposition) ofthe adipose tissue mass. In cachexia, however, the situation isdifferent. A high production of TNF by activated macrophages (as aresult of a tumor or an infection) also contributes to anorexia,increased thermogenesis, and adipose tissue dissolution. However, itrepresents a pathological state where there is an excess of themolecules that inform the brain that adipose tissue needs dissolution.The two situations can thus be reconciled: in cachexia there is apathological overproduction of TNF; in obesity, the physiological actionof TNF as a signal to control food intake and energy expenditure isimpaired. Argilés et al. (1997). FASEB J. 11:743-751.

SUMMARY OF THE INVENTION

The invention encompasses compositions of a substantially purifiedprotein having tumor necrosis factor receptor (TNFR) releasing enzymaticactivity, termed TRRE. The protein can be purified by any method knownin the art, preferably as described in the examples below. In oneembodiment, the TRRE in its native form has an apparent molecular weightof about 120 kDa on sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). In another embodiment of the presentinvention, the TRRE has internal amino acid sequences of:D-L-N-L-G-A-Q-A-T-I-T-N-L-P (SEQ ID NO:1); G-L-D-E-T-Q-N-L-I-T-V-P-Y(SEQ ID NO:2); S-E-R-W-P-Q-M-A-N-K-V-S-R (SEQ ID NO:3); I-V-V-T-K (SEQID NO:4); E-F-P-H/S-P-V-D-A-A-T-R (SEQ ID NO:5);A-L-F-E-L-I-Y-E-L-L-L/E-A-Y-I-I/N-V-L (SEQ ID NO:6);L-D-Y-Q-E/T-S-Y-S-A-A-V-A-R (SEQ ID NO:7); L-A-L-Q/I-E-S-P-S/P (SEQ IDNO:8); L-F-L-K-N-T-G-L-A-R (SEQ ID NO:9); M-A-L-Q-K-G-D-R (SEQ IDNO:10); K-L-L-E-L-N-V-V-A (SEQ ID NO:11); V/I-T-D-M-V-V-G-I-X-G (SEQ IDNO:12) where X is an unidentified amino acid residue;L-V-D-Y-D-X-L-F-Q-N-L (SEQ ID NO:13); and/or K-E-A-L-I-A-K-I-R (SEQ IDNO:14). Alternatively, in another embodiment of the present invention,the TRRE in its native form has an apparent molecular weight of about 60kDa on SDS-PAGE. In another embodiment, the TRRE has internal amino acidsequences of D-L-N-L-G-A-Q-A-T-I/L-T-N-L-P (SEQ ID NO:15);L-A-E-D-Y-L-S-G/L-W-L-E/G-R (SEQ ID NO:16); and/orL/K-V/L-D/E-Y-D/E-X-L/F-F-Q-N-L (SEQ ID NO:17). Fragments of TRREcontaining TRRE activity are also encompassed by the invention.

The invention further encompasses methods of treating a diseaseassociated with altered levels or activities of tumor necrosis factor.The methods include the steps of administering an amount of TRREsufficient to indirectly moderate or modulate local levels of tumornecrosis factor.

The invention likewise encompasses methods of treating a diseaseassociated with elevated levels of soluble TNF-R. The method includesadministering an amount of an inhibitor of TRRE effective to decreasethe levels of soluble TNF-R.

The invention also encompasses methods of diagnosing a diseaseassociated with elevated levels of TRRE, comprising obtaining abiological sample from a patient; measuring activity of TRRE in thesample and comparing the measured activity to the TRRE activity of acontrol biological sample. Excess TRRE activity compared to control isindicative of the presence of a disease state associated with elevatedlevels of TRRE.

The invention further encompasses methods of treating a diseaseassociated with decreased levels of tumor necrosis factor. The methodsinclude the steps of administering an amount of an inhibitor of TRREsufficient to modulate the levels of TNF-TNF receptor complexes on cellsurfaces.

The invention also encompasses a method for measuring TRRE activity. Themethod comprises the steps of comparing TNF-R release in TNF-Rexpressing cells (TNF-R⁺) incubated with TRRE to TNF-R release by TNF-R⁺cells not incubated with TRRE and to TNF-R release by cells that do notexpress significant amounts of TNF-R (TNF-R⁻). The amount of TNF-Rreleased by the TNF-R⁺ cells incubated with TRRE minus the amount ofTNF-R released by the TNF-R⁺ cells without TRRE and the amount of TNF-Rreleased by TNF-R⁻ cells yields the amount of TRRE in a sample.Preferably the TNF-R⁺ cells are recombinant TNF-R⁻ cells transformed toexpress TNF-R recombinantly.

The invention further encompasses methods for screening for agents thatmodulate the activity of TRRE. The activity of TREE is measured in thepresence (test) or absence (control) of a particular agent or drug. Ifthe activity of the test sample exceeds or is less than that of thecontrol, the agent or drug increases or decreases, respectively, TRREactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of plasmid pCDTR2 which expressesp75 TNF-R. PCMV stands for cytomegalovirus; BGHpA stands for bovinegrowth hormone polyadenylation signal.

FIG. 2 is a graph depicting the results of measurement of p75 TNF-R ontransfected COS-1 cells (C75R) by the method described herein. Theresults obtained with the C75R cells (●) is compared to that obtainedwith that from the parental COS-1 cells (▪). The receptor number wascalculated from a Scatchard plot (inset).

FIG. 3 is a bar graph depicting the effect of TRRE on TNF binding toC75R.

FIG. 4 depicts the results of Western Blot analysis of soluble receptorsreleased from C75R cells by TRRE.

FIG. 5 is a series of graphs depicting the time course of TNF-R sheddinginduced by TRRE. FIG. 5A depicts the short course (5-30 minutes) andFIG. 5B depicts the long course (30-90 minutes).

FIG. 6 is a graph depicting the time course of TRRE induction fromPMA-stimulated cells. The kinetics of TRRE activity and sTNF-R wereconducted on the incubation time from 3 to 24 hours including theinitial 30 min. PMA-stimulation time.

FIG. 7 is a graph depicting the effects of serial dilution of TRREmedium on production of sTNF-R.

FIG. 8 is a schematic diagram depicting the effect of successive PMAstimulations of THP-1 cells on TRRE production.

FIG. 9 depicts the induction of TRRE from THP-1 cells treated withvarious cytokines and hormones.

FIG. 10A is a bar graph depicting TRRE activity in PMA stimulated THP-1cells and controls. 10B is a photograph of gelatin zymography of thesamples listed in 10A. FIG. 10C is a bar graph depicting TRRE activityof gelatin zymography on a partially purified TRRE sample. FIG. 10D is aphotograph of gelatin zymography corresponding to the samples in 10C.

FIG. 11 is a graph depicting the TRRE activity of fractions obtainedusing a Sephadex G-150 column. Each fraction (1 ml) was assayed for TRREactivity (●) and soluble p75 TNF-R (▪), and measured for absorbance at280 nm (▾). The peak elutions of standards, beta-amylase (200 kDa) andbovine serum albumin (66 kDa) are shown.

FIG. 12A depicts the results obtained from the soluble p75 TNF-Raffinity column. Total amount of recovered TRRE from the affinity columnis adjusted to 100%. 12B depicts successive treatment of the same TRREsample to C75R cells. TRRE activity of the first treatment to C75R isadjusted to 100%.

FIG. 13 depicts the results obtained from cleavage of both p55 and p75TNF-R on THP-1 cells by TRRE.

FIG. 14 is a graph depicting the effect of TRRE against various cellsurface antigens.

FIG. 15 is a graph depicting the results of a modified in vitro TNFcytolytic assay by TRRE treatment to L929 cells.

FIG. 16 is a graph depicting the DEAE-Sephadex profile of sample Aobtained in Example 5.

FIG. 17 is a photograph of a native PAGE profile of sample B obtained inExample 5. 17A depicts the TRRE activity of each sliced strip (fraction)and 17B depicts the silver-stained native PAGE corresponding to 17A. In17B, the left side is the top of the gel.

FIG. 18 is a photograph of an SDS-PAGE of the highest TRRE eluate ofnative PAGE of sample A obtained in Example 5.

FIG. 19 is a graph depicting the effect of TRRE on preventing mortalityin mice treated with lipopolysaccharide (LPS) to induce septicperitonitis.

FIG. 20 is a bar graph demonstrating TRRE activity in human lung tumortissue (solid bar) or adjacent non-tumor tissue (white bars).

DETAILED DESCRIPTION OF THE INVENTION

TNF is a major proinflammatory and immunomodulatory cytokine producedduring immune responses. TNF also regulates the expression of IL-2Rleading to enhanced T cell responses mediated by IL-2 and appears to berequired for generating proliferative responses in mixed lymphocytecultures. Additional studies have shown that CD8⁺, CTL and lymphokineactivated killer cells are optimally induced with TNF, in combinationwith IL-2, suggesting the importance of this cytokine in regulatingcytotoxic effector function. As discussed in detail above, TNF mediatesits activity by binding to a TNF-R. Soluble TNF-Rs inhibit TNF activityby two methods: they decrease the available binding sites on a cell andbind to soluble TNF to decrease the local concentration. The presentinvention encompasses compositions and methods for modulating the levelof soluble TNF-R by decreasing the cleavage of TNF-R from the cellsurface and thus indirectly modulating the effect of TNF.

In this invention, a novel assay system was devised to detect andquantitate TRRE, resulting in the generation of sTNF-R. This proteolyticactivity, induced from PMA-stimulated THP-1 cells (a human tumormonocytic cell line) into culture medium, was defined as TNF-R releasingenzyme (TRRE). Based on this assay system, TRRE was characterized andpurified. The invention further encompasses the TRRE assay discussed inmore detail below.

The invention encompasses compositions of a substantially purifiedprotein having tumor necrosis factor receptor (TNFR) releasing enzymaticactivity, termed TRRE. The protein can be purified as described in theexamples below and, in addition to having the described enzymaticactivity, the native enzyme has an apparent molecular weight of about120 kDa on SDS-PAGE. In some embodiments of the present invention, theTRRE has internal amino acid sequences of: D-L-N-L-G-A-Q-A-T-I-T-N-L-P(SEQ ID NO:1); G-L-D-E-T-Q-N-L-I-T-V-P-Y (SEQ ID NO:2);S-E-R-W-P-Q-M-A-N-K-V-S-R (SEQ ID NO:3); I-V-V-T-K (SEQ ID NO:4);E-F-P-H/S-P-V-D-A-A-T-R (SEQ ID NO:5);A-L-F-E-L-I-Y-E-L-L-L/E-A-Y-I-I/N-V-L (SEQ ID NO:6);L-D-Y-Q-E/T-S-Y-S-A-A-V-A-R (SEQ ID NO:7); L-A-L-Q/I-E-S-P-S/P (SEQ IDNO:8); L-F-L-K-N-T-G-L-A-R (SEQ ID NO:9); M-A-L-Q-K-G-D-R (SEQ IDNO:10); K-L-L-E-L-N-V-V-A (SEQ ID NO:11); V/I-T-D-M-V-V-G-I-X-G (SEQ IDNO:12) where X is an unidentified amino acid residue;L-V-D-Y-D-X-L-F-Q-N-L (SEQ ID NO:13); and K-E-A-L-I-A-K-I-R (SEQ IDNO:14). Alternatively, the TRRE in its native form has an apparentmolecular weight of about 60 kDa on SDS-PAGE. In some embodiments of thepresent invention, the TRRE has internal amino acid sequences ofD-L-N-L-G-A-Q-A-T-I/L-T-N-L-P (SEQ ID NO:15);L-A-E-D-Y-L-S-G/L-W-L-E/G-R (SEQ ID NO:16); andL/K-V/L-D/E-Y-D/E-X-L/F-F-Q-N-L (SEQ ID NO:17) where X is anunidentified amino acid residue. The banding pattern on SDS-PAGE issomewhat diffuse, indicating that the protein may be a glycoprotein.Thus, recombinant proteins (i.e. “non-native” proteins) may havediffering apparent molecular weights depending on the degree ofglycosylation.

The enzymatic activity of the TRRE is inhibited by metalloproteaseinhibitors. Thus, the method of inhibiting the activity of TRRE by theaddition of metalloprotease inhibitors is also encompassed by theinvention.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acid residues ofany length. The polymer may be linear or branched, it may comprisemodified amino acids or amino acid analogs, and it may be interrupted bychemical moieties other than amino acids. The terms also encompass anamino acid polymer that has been modified naturally or by intervention;for example, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling or bioactive component. Unlessstated or implied otherwise, the term TRRE includes any polypeptidemonomer or polymer with TRRE enzymatic specificity, including the intactTRRE, and smaller and larger functionally equivalent polypeptides, asdescribed herein.

A “fusion polypeptide” is a polypeptide comprising regions in adifferent position in the sequence than occurs in nature. The regionsmay normally exist in separate proteins and are brought together in thefusion polypeptide; they may normally exist in the same protein but areplaced in a new arrangement in the fusion polypeptide; or they may besynthetically arranged. For instance, as described below, the inventionencompasses recombinant proteins that are comprised of a functionalportion of TRRE and an antibody. Methods of making these fusion proteinsare known in the art and are described, for instance, in W093/07286.

A “functionally equivalent fragment” of a TRRE polypeptide varies fromthe native sequence by addition(s), deletion(s), or substitution(s), orany combination thereof, while preserving at least one functionalproperty of the fragment relevant to the context in which it is beingused. A functionally equivalent fragment of a TRRE polypeptide typicallyhas the ability to bind membrane bound TNF-R and enzymatically cleaveTNF-R to provide soluble TNF-R. Amino acid substitutions, if present,are preferably conservative substitutions that do not deleteriouslyaffect folding or functional properties of the peptide. Groups offunctionally related amino acids within which conservative substitutionscan be made are glycine/alanine; valine/isoleucine/leucine;asparagine/glutamine; aspartic acid/glutamic acid;serine/threonine/methionine; lysine/arginine; andphenylalanine/tyrosine/tryptophan. Polypeptides of this invention can bein glycosylated or unglycosylated form, can be modifiedpost-translationally (e.g., removal of signal peptide, transmembrane orcytoplasmic regions, acetylation, and phosphorylation) or can bemodified synthetically (e.g., by a labeling group).

The invention also encompasses compositions of TRRE and aphysiologically acceptable buffer. Suitable physiologically acceptablebuffers include, but are not limited to, saline and phosphate bufferedsaline (PBS). If TRRE is to be administered to an individual, it ispreferably at least 80% pure, more preferably it is at least 90% pure,even more preferably it is at least 95% pure and free of pyrogens andother contaminants. In this context, the percent purity is calculated asa weight percent of the total protein content of the preparation, anddoes not include constituents which are deliberately added to thecomposition after the TRRE is purified.

The invention further encompasses antibodies (or antigen-bindingfragments thereof) specific for TRRE proteins. The compositions containa therapeutically effective amount of substantially purified antibodybinding fragment specific for TRRE. Preferably, the antibody neutralizesTRRE TNF-R releasing activity. Methods of antibody production andisolation are well known in the art. See, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York. Antibodies can be isolated by any technique suitable forimmunoglobulins of this isotype. Purification methods include saltprecipitation (for example, with ammonium sulfate), ion exchangechromatography (for example, on a cationic or anionic exchange columnrun at neutral pH and eluted with step gradients of increasing ionicstrength), gel filtration chromatography (including gel filtrationHPLC), and chromatography on affinity resins such as protein A, proteinG, hydroxyapatite, and anti-immunoglobulin. Anti-TRRE antibodies canalso be purified on affinity columns comprising TRRE. Preferably,anti-TRRE antibodies are purified using Protein-A-CL-Sepharos™ 4Bchromatography followed by chromatography on a DEAE-Sepharose™ 4B ionexchange column.

The term “antigen-binding fragment” includes any peptide that binds toTRRE in a specific manner. These derivatives include such immunoglobulinfragments as Fab, F(ab′)₂, Fab′, scfv (both monomers and polymericforms) and isolated H and L chains. Antigen-binding fragments retain thespecificity of the intact immunoglobulin, although avidity and/oraffinity can be altered.

The antigen-binding fragments (also termed “derivatives” herein) aretypically generated by genetic engineering, although they canalternatively be obtained by other methods and combinations of methods.This classification includes, but is not limited to, engineered peptidefragments and fusion peptides. Preferred compounds include polypeptidefragments of the CDRs, antibody fusion proteins comprising cytokineeffector components, antibody fusion proteins comprising adjuvants ordrugs, and single-chain V region proteins.

Scfv can be produced either recombinantly or synthetically. Forsynthetic production of scfv, an automated synthesizer can be used. Forrecombinant production of scfv, a suitable plasmid containingpolynucleotide that encodes the scfv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as E. coli, and the expressed protein can beisolated using standard protein purification techniques.

A particularly useful system for the production of scfvs is plasmidpET-22b(+) (Novagen, Madison, Wis.) in E. coli. pET-22b(+) contains anickel ion binding domain consisting of 6 sequential histidine residues,which allows the expressed protein to be purified on a suitable affinityresin. Another example of a suitable vector is pcDNA3 (Invitrogen, SanDiego, Calif.), described above.

The invention also encompasses hybrid antibodies, in which one pair of Hand L chains is obtained from a first antibody, while the other pair ofH and L chains is obtained from a different second antibody. Forpurposes of this invention, one pair of L and H chains is fromanti-TRRE. In one example, each L-H chain pair binds different epitopesof TRRE. Such hybrids can also be formed using humanized H or L chains.Also encompassed by the invention are peptides in which variousimmunoglobulin domains have been placed in an order other than thatwhich occurs in nature. Additionally, the antigen-binding fragments ofthis invention can be used as diagnostic and imaging reagents.

The invention further encompasses polynucleotides encoding antibodies(or fragments thereof) capable of binding to a TRRE polypeptide (orfragments thereof). The polynucleotide can be native or recombinant, andcan be incorporated into a vector or plasmid and operably linked to apromoter.

The invention further encompasses methods of treating a diseaseassociated with altered levels or activities of TNF by administering anamount of TRRE sufficient to indirectly reduce local levels of TNF.Suitable indications for treatment include, but are not limited to,diseases such as cancer, obesity and cachexia, autoimmune diseases suchas diabetes, juvenile onset rheumatoid arthritis, systemic lupuserythematosus, and other inflammatory conditions, psoriasis, endotoxinshock, rheumatoid arthritis, trauma, and multiple sclerosis. Infectionsassociated with microbial or parasitic infection in which TNF has animportant role include, but are not limited to, septic shock andmalaria. In addition, TRRE can be used to treat indications previouslyassociated with aberrant MMP expression. As mentioned above, theseinclude, but are not limited to, angiogenesis; wound healing; gumdisease; skin disorders; keratoconus; inflammatory conditions;rheumatoid arthritis; cancer; corneal and skin ulcers; cardiovasculardisease; central nervous system disorders; and diabetes. Also includedare immuno-inflammatory diseases or diseases with an immunological baseand a tissue destructive component in which TNF has an important rolesuch as Crohn's disease and inflammatory bowel disease. Otherinflammatory conditions such as traumatic shock are also included.

Methods of administration include any known in the art. Preferably,administration is directly to the site of inflammation in the case ofrheumatoid arthritis and is parenteral, subcutaneous, intramuscular,intraperitoneal, intracavity, intrathecal, and intravenous in the caseof systemic disorders. Administration can also be systemic to treatlocalized disorders or local to treat systemic disorders. Methods ofadministration are discussed in more detail below. Local administrationcan be achieved by, for example, local infusion during surgery, bydirect injection to the site, by means of a catheter, or by means of animplant, said implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as silastic membranes, or fibers. Asuitable such membrane is Gliadel® provided by Guilford sciences.

With external ailments such as psoriasis, treatment is by topicalapplication. The invention thus further includes compositions ofeffective amounts of TRRE and a topical pharmaceutically or cosmeticallyacceptable carrier.

“Topical pharmaceutically acceptable carrier” as used herein is anysubstantially non-toxic carrier conventionally useable for topicaladministration of pharmaceuticals in which TRRE will remain stable andbioavailable when applied directly to skin or mucosal surfaces. Forexample, TRRE can be dissolved in a liquid, dispersed or emulsified in amedium in a conventional manner to form a liquid preparation or mixedwith a semi-solid (gel) or solid carrier to form a paste, powder,ointment, cream, lotion or the like.

Suitable topical pharmaceutically acceptable carriers include water,petroleum jelly (Vaseline™), petrolatum, mineral oil, vegetable oil,animal oil, organic and inorganic waxes, such as microcrystalline,paraffin and ozocerite wax, natural polymers, such as xanthanes,gelatin, cellulose, collagen, starch, or gum arabic, synthetic polymers,such as discussed below, alcohols, polyols, and the like. The carriercan be a water miscible carrier composition that is substantiallymiscible in water. Such water miscible topical pharmaceuticallyacceptable carrier composition can include those made with one or moreappropriate ingredients set forth above but can also include sustainedor delayed release carriers, including water containing, waterdispersible or water soluble compositions, such as liposomes,microsponges, microspheres or microcapsules, aqueous base ointments,water-in-oil or oil-in-water emulsions, gels or the like.

In one embodiment of the invention, the topical pharmaceuticallyacceptable carrier comprises a sustained release or delayed releasecarrier. The carrier is any material capable of sustained or delayedrelease of the TRRE to provide a more efficient administration resultingin one or more of less frequent and/or decreased dosage of the TRRE,ease of handling, and extended or delayed effects on dermatologicconditions. The carrier is capable of releasing TRRE when exposed to anyoily, fatty, waxy, or moist environment on the area being treated or bydiffusing or by release dependent on the degree of loading of TRRE tothe carrier in order to obtain release of TRRE. Non-limiting examples ofsuch carriers include liposomes, microsponges, microspheres, ormicrocapsules of natural and synthetic polymers and the like. Examplesof suitable carriers for sustained or delayed release in a moistenvironment include gelatin, gum arabic; xanthane polymers; by degree ofloading include lignin polymers and the like; by oily, fatty or waxyenvironment include thermoplastic or flexible thermoset resin orelastomer including thermoplastic resins such as polyvinyl halides,polyvinyl esters, polyvinylidene halides and halogenated polyolefins,elastomers such as brasiliensis, polydienes, and halogenated natural andsynthetic rubbers, and flexible thermoset resins such as polyurethanes,epoxy resins and the like. Preferably, the sustained or delayed releasecarrier is a liposome, microsponge, microsphere or gel.

The compositions used in the method of treating dermatologic conditionsof the invention are applied directly to the areas to be treated. Whilenot required, it is desirable that the topical composition maintain TRREat the desired location for about 24 to 48 hours.

If desired, one or more additional ingredients conventionally found intopical pharmaceutical or cosmetic compositions can be included with thecarrier, such as a moisturizer, humectants, odor modifier, buffer,pigment, preservative, Vitamins such as A, C and E, emulsifier,dispersing agent, wetting agent, odor-modifying agent, gelling agents,stabilizer, propellant, antimicrobial agents, sunscreen, enzymes and thelike. Those of skill in the art of topical pharmaceutical formulationscan readily select the appropriate specific additional ingredients andamounts thereof. Suitable non-limiting examples of additionalingredients include superoxide dismutase, stearyl alcohol, isopropylmyristate, sorbitan monooleate, polyoxyethylene stearate, propyleneglycol, water, alkali or alkaline earth lauryl sulfate, methylparaben,octyl dimethyl-p-amino benzoic acid (Padimate O), uric acid, reticulin,polymucosaccharides, hyaluronic acids, aloe vera, lecithin,polyoxyethylene sorbitan monooleate, Vitamin A or C, tocopherol (VitaminE), alpha-hydroxy of alpha-keto acids such as pyruvic, lactic orglycolic acids, or any of the topical ingredients disclosed in U.S. Pat.Nos. 4,340,586, 4,695,590, 4,959,353 or 5,130,298 and 5,140,043.

Because dermatologic conditions to be treated can be visible, thetopical carrier can also be a topical cosmetically acceptable carrier.By “topical cosmetically acceptable carrier” as used herein is meant anysubstantially non-toxic carrier conventionally useable for topicaladministration of cosmetics in which TRRE will remain stable andbioavailable when applied directly to the skin surface. Suitablecosmetically acceptable carriers are known to those of skill in the artand include, but are not limited to, cosmetically acceptable liquids,creams, oils, lotions, ointments, gels, or solids, such as conventionalcosmetic night creams, foundation creams, suntan lotions, sunscreens,hand lotions, make-up and make-up bases, masks and the like. Thus, to asubstantial extent, topical cosmetically acceptable carriers andpharmaceutically acceptable carriers are similar, if not oftenidentical, in nature so that most of the earlier discussion onpharmaceutically acceptable carriers also applies to cosmeticallyacceptable carriers. The compositions can contain other ingredientsconventional in cosmetics including perfumes, estrogen, Vitamins A, C orE, alpha-hydroxy or alpha-keto acids such as pyruvic, lactic or glycolicacids, lanolin, Vaseline™ petroleum jelly, aloe vera, methyl or propylparaben, pigments and the like.

The effective amount of TRRE in the compositions used to treatdermatologic conditions or diseases can vary depending on such factorsas condition of the skin, age of the skin, the degree of purity of TRREemployed, the type of formulation and carrier ingredients used,frequency of administration, overall health of the individual beingtreated and the like. The precise amount for any particular patient usedcan be determined by those of skill in the pharmaceutical art takinginto consideration these factors and the present disclosure. Preferablythe composition is administered in at least two doses and no more thanabout six doses per day, or less when a sustained or delayed releaseform is used.

The compositions for topical, oral and parenteral administration usuallycontain from about 0.001% to about 10% by weight of TRRE compared to thetotal weight of the composition, preferably from about 0.01% to about 2%by weight of TRRE to composition, and especially from about 0.1% toabout 1.5% by weight of TRRE to the composition.

The topical composition is administered by applying a coating or layerto the skin or mucosal area desired to be treated. As a practical matterof convenience, the applied material is rubbed into the area.Applications need not be rubbed into the skin and the layer or coatingcan be left on the skin overnight.

The amount of TRRE administered is sufficient to effectively reducelevels of TNF. Typically, reduced levels of TNF result in anamelioration of symptoms. Ameliorate denotes a lessening of thedetrimental effect of the cancer on the individual. Administrations aretypically conducted on a weekly or biweekly basis until a desired,measurable parameter is detected, such as diminution of diseasesymptoms. Administration can then be continued on a less frequent basis,such as biweekly or monthly, as appropriate. The effective amount isreadily determined by one of skill in the art. The dosage ranges arethose large enough to produce the desired effect in which the symptomsof the disease are ameliorated without causing undue side effects suchas unwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any complication.

The invention further encompasses methods of treating a diseaseassociated with elevated levels of soluble TNF-R by administering anamount of an inhibitor of TRRE effective to decrease the levels ofsoluble TNF-R. Preferably, the disease treated is cancer. Morepreferably, the cancer includes, but is not limited to, astrocytoma,oligodendroglioma, ependymoma, medulloblastoma, primitive neuralectodermal tumor, pancreatic ductal adenocarcinoma, small and large celllung adenocarcinomas, squamous cell carcinoma, bronchoalveolarcarcinoma,epithelial adenocarcinoma and liver metastases thereof, hepatoma,cholangiocarcinoma, ductal and lobular adenocarcinoma, squamous andadenocarcinomas of the uterine cervix, uterine and ovarian epithelialcarcinomas, prostatic adenocarcinomas, transitional squamous cellbladder carcinoma, B and T cell lymphomas (nodular and diffuse),plasmacytoma, acute and chronic leukemias, malignant melanoma, softtissue sarcomas and leiomyosarcomas.

The TRRE inhibitor can be any known in the art, including, but notlimited to, metalloprotease inhibitors, antibodies that block theeffective interaction between TNF-R and TRRE, antisense oligonucleotidesspecific for the gene encoding TRRE and ribozymes specific for the geneencoding TRRE. Methods of making antibodies are known in the art.Antibodies that block effective binding of TRRE to the TNF-R are easilyscreened for by using the assay method described herein.

The dosage ranges for the administration of TRRE inhibitors are thoselarge enough to produce the desired effect in which the symptoms of themalignant disease are ameliorated without causing undue side effectssuch as unwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any complication. For instance, when the inhibitor is anantibody, dosage can vary from about 0.1 mg/kg to about 2000 mg/kg,preferably about 0.1 mg/kg to about 500 mg/kg, in one or more doseadministrations daily, for one or several days. Generally, when theantibodies are administered conjugated with therapeutic agents, lowerdosages, can be used.

Therapeutic compositions of TRRE inhibitors can be administered byinjection or by gradual perfusion over time. The inhibitors can beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, intrathecally or transdermally, alone or incombination with effector cells.

Preferably, in the case of cancer treatment, administration isintralesionally, for instance by direct injection directly into thetumor. Intralesional administration of various forms of immunotherapy tocancer patients does not cause the toxicity seen with systemicadministration of immunologic agents. Fletcher et al. (1987) LymphokineRes. 6:45; Rabinowich et al. (1987) Cancer Res. 47:173; Rosenberg et al.(1989) Science 233:1318; and Pizz et al (1984) Int. J. Cancer 34:359.Preferably, the intralesional administration is in conjunction with thecancer therapy technology described in, for example, U.S. Pat. Nos.5,376,682, 5,192,537, and 5,643,740.

When the site of delivery is the brain, the therapeutic agent must becapable of being delivered to the brain. The blood-brain barrier limitsthe uptake of many therapeutic agents into the brain and spinal cordfrom the general circulation. Molecules which cross the blood-brainbarrier use two main mechanisms: free diffusion; and facilitatedtransport. Because of the presence of the blood-brain barrier, attainingbeneficial concentrations of a given therapeutic agent in the CNS mayrequire the use of drug delivery strategies. Delivery of therapeuticagents to the CNS can be achieved by several methods.

One method relies on neurosurgical techniques. In the case of gravelyill patients, surgical intervention is warranted despite its attendantrisks. For instance, therapeutic agents can be delivered by directphysical introduction into the CNS, such as intraventricular,intralesional, or intrathecal injection. Intraventricular injection canbe facilitated by an intraventricular catheter, for example, attached toa reservoir, such as an Ommaya reservoir. Methods of introduction canalso be provided by rechargeable or biodegradable devices. Anotherapproach is the disruption of the blood-brain barrier by substanceswhich increase the permeability of the blood-brain barrier. Examplesinclude intra-arterial infusion of poorly diffusible agents such asmannitol, pharmaceuticals which increase cerebrovascular permeabilitysuch as etoposide, or vasoactive agents such as leukotrienes. Neuweltand Rappoport (1984) Fed. Proc. 43:214-219; Baba et al. (1991) J. Cereb.Blood Flow Metab. 11:638-643; and Gennuso et al. (1993) Cancer Invest.11:638-643.

Further, it may be desirable to administer the compositions locally tothe area in need of treatment; this can be achieved by, for example,local infusion during surgery, by injection, by means of a catheter, orby means of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as silastic membranes, orfibers. A suitable such membrane is Gliadel® provided by Guilfordsciences.

Another method involves pharmacological techniques such as modificationor selection of the inhibitor to provide an analog which will cross theblood-brain barrier. Examples include increasing the hydrophobicity ofthe molecule, decreasing net charge or molecular weight of the molecule,or modifying the molecule, such as to resemble one normally transportedacross the blood-brain barrier. Levin (1980) J. Med. Chem. 23:682-684;Pardridge (1991) in: Peptide Drug Delivery to the Brain; and Kostis etal. (1994) J. Clin. Pharmacol. 34:989-996.

Encapsulation of an inhibitor in a hydrophobic environment such asliposomes is also effective in delivering drugs to the CNS. For example,WO 91/04014 describes a liposomal delivery system in which the drug isencapsulated within liposomes to which molecules have been added thatare normally transported across the blood-brain barrier.

Another method of formulating the inhibitors to pass through theblood-brain barrier is encapsulation in cyclodextrin. Any suitablecyclodextrin which passes through the blood-brain barrier can beemployed, including, but not limited to, β-cyclodextrin, γ-cyclodextrinand derivatives thereof. See generally, U.S. Pat. Nos. 5,017,566,5,002,935 and 4,983,586. Such compositions can also include a glycerolderivative as described by U.S. Pat. No. 5,153,179.

Yet another method takes advantage of physiological techniques such asconjugation of the inhibitor to a transportable agent to yield a newchimeric transportable αC. For example, vasoactive intestinal peptideanalog (VIPa) exerted its vasoactive effects only after conjugation to aMab to the specific carrier molecule transferrin receptor, whichfacilitated the uptake of the VIPa-Mab conjugate through the blood-brainbarrier. Pardridge (1991); and Bickel et al. (1993) Proc. Natl. Acad.Sci. USA 90:2618-2622. Several other specific transport systems havebeen identified, these include, but are not limited to, those fortransferring insulin, or insulin-like growth factors I and II. Othersuitable, non-specific carriers include, but are not limited to,pyridinium, fatty acids, inositol, cholesterol, and glucose derivatives.Certain prodrugs have been described whereby, upon entering the centralnervous system, the drug is cleaved from the carrier to release theactive drug. U.S. Pat. No. 5,017,566.

The TRRE inhibitor can be administered in conjunction with at least onecytokine effective to enhance an immune response against the cancer.Suitable cytokines include, but are not limited to, TNF, interleukin 2(IL-2), interleukin 4 (IL-4), granulocyte macrophage colony stimulatingfactor (GM-CSF), and granulocyte colony stimulating factor (GCSF).

The TRRE inhibitor can be further administered in conjunction with atleast one chemotherapeutic agent. Suitable chemotherapeutic agentsinclude, but are not limited to, radioisotopes, vinca alkaloids,adriamycin, bleomycin sulfate, Carboplatin, cisplatin, cyclophosphamide,Cytarabine, Dacarbazine, Dactinomycin, Duanorubicin hydrochloride,Doxorubicin hydrochloride, Etoposide, fluorouracil, lomustine,mechlororethamine hydrochloride, melphalan, mercaptopurine,methotrexate, mitomycin, mitotane, pentostatin, pipobroman, procarbazehydrochloride, streptozotocin, taxol, thioguanine, and uracil mustard.

Suitable subjects include those who are suspected of being at risk of apathological effect of any neoplasia, particularly carcinoma, aresuitable for treatment with the pharmaceutical compositions of thisinvention. Those with a history of cancer are especially suitable.Suitable human subjects for therapy comprise two groups, which may bedistinguished by clinical criteria. Patients with “advanced disease” or“high tumor burden” are those who bear a clinically measurable tumor. Aclinically measurable tumor is one that can be detected on the basis oftumor mass (e.g., by palpation, CAT scan, or X-ray; positive biochemicalor histopathological markers on their own are insufficient to identifythis population). A pharmaceutical composition embodied in thisinvention is administered to these patients to elicit an anti-tumorresponse, with the objective of palliating their condition. Ideally,reduction in tumor mass occurs as a result, but any clinical improvementconstitutes a benefit. Clinical improvement includes decreased risk orrate of progression or reduction in pathological consequences of thetumor.

A second group of suitable subjects is known in the art as the “adjuvantgroup”. These are individuals who have had a history of cancer, but havebeen responsive to another mode of therapy. The prior therapy may haveincluded (but is not restricted to) surgical resection, radiotherapy,and traditional chemotherapy. As a result, these individuals have noclinically measurable tumor. However, they are suspected of being atrisk for progression of the disease, either near the original tumorsite, or by metastases.

This group may be further subdivided into high-risk and low-riskindividuals. The subdivision is made on the basis of features observedbefore or after the initial treatment. These features are known in theclinical arts, and are suitably defined for each different cancer.Features typical of high risk subgroups are those in which the tumor hasinvaded neighboring tissues, or who show involvement of lymph nodes.

Another suitable group of subjects is those with a geneticpredisposition to cancer but who have not yet evidenced clinical signsof cancer. For instance, women testing positive for a genetic mutationassociated with breast cancer, but still of childbearing age, may wishto receive TRRE inhibitor treatment prophylactically to prevent theoccurrence of cancer until it is suitable to perform preventive surgery.

A pharmaceutical TRRE inhibitor composition embodied in this inventionis administered to patients in the adjuvant group, or in either of thesesubgroups, in order to elicit an anti-cancer response. Ideally, thecomposition delays recurrence of the cancer, or even better, reduces therisk of recurrence (i.e., improves the cure rate). Such parameters maybe determined in comparison with other patient populations and othermodes of therapy.

Of course, crossovers between these two patient groups occur, and thepharmaceutical compositions of this invention can be administered at anytime that is appropriate. For example, therapy can be conducted beforeor during traditional therapy of a patient with high tumor burden, andcontinued after the tumor becomes clinically undetectable. Therapy canbe continued in a patient who initially fell in the adjuvant group, butis showing signs of recurrence. The attending physician can determinehow or when the compositions of this invention are to be used.

The invention also encompasses methods of diagnosing a diseaseassociated with elevated levels of TRRE by obtaining a biological samplefrom a patient; measuring activity of TRRE in the sample and comparingthe activity to the activity of a control biological sample. In the caseof cancer diagnosis, the increased level of TRRE activity compared tothe control can indicate that cancer is present. In the case ofmonitoring progression or recurrence of the disease, measurement of TRREcan indicate the status of the disease and can be an early marker forrecurrence.

As provided herein, treatment, diagnosis and monitoring of cancersincludes any cancers known in the art. These include, but are notlimited to, glioblastoma, melanoma, neuroblastoma, adenocarcinoma, softtissue sarcoma, leukemias, lymphomas and carcinoma. The invention isparticularly useful for treatment, diagnosis and monitoring ofcarcinomas. Carcinomas include, but are not limited to, astrocytoma,oligodendroglioma, ependymoma, medulloblastoma, primitive neuralectodermal tumor, pancreatic ductal adenocarcinoma, small and large celllung adenocarcinomas, squamous cell carcinoma, bronchoalveolarcarcinoma,epithelial adenocarcinoma and liver metastases thereof, hepatoma,cholangiocarcinoma, ductal and lobular adenocarcinoma, squamous andadenocarcinomas of the uterine cervix, uterine and ovarian epithelialcarcinomas, prostatic adenocarcinomas, transitional squamous cellbladder carcinoma, B and T cell lymphomas (nodular and diffuse),plasmacytoma, acute and chronic leukemias, malignant melanoma, softtissue sarcomas and leiomyosarcomas.

Embodied in this invention are compositions comprising polynucleotideswith a therapeutically relevant genetic sequence as an activeingredient. The polynucleotide can be administered, for example, toaugment or attenuate the natural level of expression of TRRE within atarget cell.

In a another approach to attenuate TRRE activity, a polynucleotideencodes an antibody (or a fragment thereof) capable to binding to TRRE(or a fragment thereof). The polynucleotide would be introduced into thecell, and then expressed to produce the antibody or antibody fragment,which would then act as described supra to bind to TRRE and modulate itsactivity.

A polynucleotide for enhancing or attenuating TRRE expression can beintroduced into cells as part of any suitable delivery vehicle known inthe art. The polynucleotide can be administered to cells or injectedinto a tissue site as naked DNA, preferably in a supercoiledconfiguration. It is generally preferred to administer thepolynucleotide as part of a composition that enhances expression in thetarget cell. Components of the composition can include those thatprotect the polynucleotide until delivery to the cell, enhance bindingto or localization near target cells, enhance uptake or endocytosis intocells, promote translocation of the polynucleotide across the membraneinto the cytoplasm, or enhance transport of the polynucleotide insidethe cell to the site of action.

In one example, the composition comprises one half of a ligand-receptorbinding pair, the other of which is present on the surface of the targetcell. This can promote localization near the cell surface, endocytosisinto the cell, or homing to the cell in vivo, or any combinationthereof. Suitable components for including in the composition include,but are not limited to, antibodies or antibody fragments specific forthe target tissue (for example, a tumor-associated antigen), integrinsand integrin ligands optionally specific for the target tissue, andligands for cytokine receptors on the target tissue. Where the object isto decrease TNF-R levels on the target cell by enhancing TRREexpression, a particularly preferred ligand is TNF itself. In this way,the composition will be focused towards cells with the phenotype to betreated, in preference to other cell types and cells already treatedeffectively.

In another example, the composition comprises a delivery vehicle thatprotects the polynucleotide and enhances its delivery into the cell. Onetype of suitable vehicle is a liposome that either encapsulates thepolynucleotide, or (in the case of cationic liposomes) binds it bycharge association. Another type of suitable vehicle is the capsid orenvelope of a virus, defective viral particle, or synthetic viralparticle, encapsidating or enveloping the polynucleotide. Preferredamongst such virally related particles are those that are tropic for thetarget tissue type, and comprise polypeptides (such as the influenzahemagglutinin) that promote fusion and delivery of the polynucleotide.The composition can also optionally comprise genetic elements of a virusthat promotes replication of the therapeutic polynucleotide and/orintegration into the genome of the target cell. Suitable viral systemsfor use with this invention include adenovirus vectors, retroviralvectors, adeno-associated viral vectors, sindbis virus vectors, and thelike. Preferred are vectors that comprise viral genetic elementsrequired in cis for packaging, the genetic elements required forreplication or integration of the therapeutic polynucleotide, but notother viral genetic elements. Such vectors can be produced by packagingsystems in which viral elements required only in trans are supplied by ahost cell or second virus. See, e.g., Flotte et al. WO 95/13365.

It is often preferable to combine several such components and strategiesinto the composition with the therapeutic polynucleotide. For example, apolynucleotide can be enveloped in an adenovirus vector that expresses atargeting molecule like TNF as part of the viral package. The vectormight alternatively express a coupling molecule, such as an avidinbinding site, that can then be coupled with biotin-TNF for purposes oftargeting to the target cell.

The following examples are meant to illustrate, but not limit, theclaimed invention.

EXAMPLE 1 Materials and Methods

Cell Lines & Reagents:

COS-1, a monkey kidney fibroblast-like cell line, was obtained fromAmerican Type Culture Collection (ATCC) (Rockville, Md.) were maintainedas an adherent monolayer in RPMI-1640 medium (GIBCO Laboratories, GrandIsland, N.Y.) supplemented with heat inactivated 10% fetal calf serum(FCS) (Irvine Scientific, Santa Ana, Calif.) and passed twice weekly.The cells were maintained at 37° C. in a humidified atmosphere of 5%CO₂. The transfected COS-1 cell line termed C75R was maintained in thesame medium with the addition of 600 μg/ml Geneticin (G418-sulfate)(GIBCO BRL Life Technologies, Gaithersburg, Md.) and passed twiceweekly. THP-1, a human monocytic leukemia cell line, was obtained fromATCC and maintained as a suspension culture in RPMI-1640 supplementedwith 10% heat-inactivated FCS. PMA was purchased from Sigma Chemical Co.(St. Louis, Mo.). Recombinant forms of human soluble p55 and p75 TNF-Rand TNF were kindly provided by Synergen Inc. (Boulder, Colo.).

ELISA assays were performed by the following methods. Polyclonalantibodies to human p75 TNF-R were generated by immunization of NewZealand white female rabbits according to the techniques described byYamamoto et al. (1978) Cell. Immunol. 38:403-416. The IgG fraction ofthe immunized rabbit serum was purified using a protein G (PharmaciaFine Chemicals, Uppsala, Sweden) affinity column by the method of Ey etal. (1978) Immunochemistry 15:429-436. The IgG fraction was then labeledwith horseradish peroxidase (Sigma Chemical Co., St. Louis, Mo.) asdescribed by Tijssen and Kurstok (1984) Anal. Biochem. 136:451-457.

The ELISA for p75 TNF-R was performed as follows: first, 5 μg ofunlabeled IgG in 100 μl of 0.05 M carbonate buffer (pH 9.6) was bound toa 96-well ELISA microplate (Corning, Corning, N.Y.) by overnightincubation at 4° C. Individual wells were washed three times with 300 μlof 0.2% Tween-20 in phosphate buffered saline (PBS). The 100 μl ofsamples and recombinant receptor standards were then added to each welland incubated at 37° C. for 1 to 2 hours. The wells were then washed inthe same manner, 100 μl of horseradish peroxidase-labeled IgG added andincubated for 1 hour at 37° C. The wells were washed once more and thecolor was developed for 20 minutes (min) at room temperature with thesubstrate of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic aciddiammonium salt (ABTS) (Pierce, Rockford, Ill.) and 30% H₂O₂ (FisherScientific, Fair Lawn, N.J.) prepared according to manufacturer'sinstructions. The results were obtained by measuring the absorbance at405 nm using an EAR 400AT plate reader (SLT-Lab Instruments, Salzburg,Austria). The concentration of sTNF-R in each sample was calculated fromthe regression line computed by known standards. Most of the R2 valuesof the linear regression were greater than 0.99. Duplicate wells of eachdilution or sample were tested and the average of the results wasreported.

In order to induce secretion of TRRE from THP-1 cells, the followingexperiment was performed. THP-1 cells growing in logarithmic phase werecollected and resuspended to 1×10⁶ cells/ml of RPMI-1640 supplementedwith 1% FCS and incubated with 10⁻⁶ M PMA for 30 min in 5% CO₂ at 37° C.The cells were collected and washed once with serum-free medium toremove PMA and resuspended in the same volume of RPMI-1640 with 1% FCS.After 2 hours incubation in 5% CO₂ at 37° C., the cell suspension wascollected, centrifuged, and the cell-free supernatant was collected asthe TRRE-containing sample.

The level of TRRE activity in the THP-1 supernatant collected wasdetected and quantitated as described and was measured with a novelassay system described herein in detail below. Briefly, C75R and COS-1cells were seeded at 2.5×10⁵ cells/ml/well in a 24-well culture plateand incubated in 5% CO₂ at 37° C. for 12 to 16 hours. The medium in thewells was aspirated and 300 μl of TRRE experimental sample was incubatedwith C75R and COS-1 cells for 30 min in 5% CO₂ at 37° C. Simultaneously,C75R was incubated with 300 μl of fresh medium or buffer. At the end ofthe incubation period, the samples from each well were collected and thelevels of soluble p75 TNF-R were measured by ELISA. The background levelof sTNF-R, which was measured by incubation of the TRRE experimentalsample with COS-1, and the spontaneous release of receptors by C75R,which was measured by incubation of medium or buffer alone with C75Rcells, were subtracted from the level measured by the TRRE experimentalsample incubated with C75R in order to calculate the net TRRE activity.

EXAMPLE 2 In Vitro TRRE Assay System

The objective of this study was to establish an assay system thatmeasures TRRE activity on the human TNF-R in its native conformationintegrated into the cell surface membrane. The transfected COS-1 cellline was chosen for the assay system since no background of endogenousp75 TNF-R was observed. Attempts to study and characterize the enzymeresponsible for sTNF-R release have been difficult because the presenceof an active form of the proteolytic enzyme is indicated only indirectlyby the generation of soluble receptors. Studies of release of othermembrane bound proteins as well as TNF-R have been carried out bymeasuring the levels of soluble counterparts by ELISA or by FACSanalysis for the presence or absence of the surface antigens. Therefore,the level of the enzyme itself has not yet been quantitated. Wetherefore devised a novel assay system to detect and quantitate TRRE. Itwas found that the level of soluble forms released into the mediumdepends on the level of expression of surface antigens on the membraneand the rate at which the cells can synthesize more and express theseproteins on the membrane. In some studies, the enzyme levels and thekinetics of active enzyme formed have been correlated with the levels ofsoluble forms released and the kinetics of their release. We have nowdevised a more defined assay system to detect and also quantitate TRREspecifically and enzymes that cleave membrane receptor proteins ingeneral.

Membrane-associated TNF-R was chosen as the substrate for TRRE insteadof the recombinant TNF-R molecule, because the membrane-associated TNF-Rsimulates a more physiological microenvironment and substrate for theevaluation of TRRE activity. Membrane-associated TNF-R can also assistin alleviating nonspecific cleavage by other proteases which can occurin nonmembrane-associated forms. Since most human cells express onlyextremely low levels of both TNF-Rs, human p75 TNF-R-overexpressingcells were constructed by cDNA transfection into monkey COS-1 cellswhich do not express either TNF-Rs.

The cDNA of the human p75 TNF-R was cloned from a λgt10 cDNA libraryderived from human monocytic U-937 cells (Clonetech Laboratories, PaloAlto, Calif.). The cDNA was then subcloned into the EcoRI site of themammalian expression vector pCDNA3 (Invitrogen, San Diego, Calif.) whichcontains the neomycin-resistance gene for the selection of transfectedcells in the presence of G418. This construct was transfected into COS-1cells using the calcium phosphate-DNA precipitation method described byChen and Okayama. 24 hours post transfection, the transfected cells wereplaced in 600 μg/ml G418 (GIBCO BRL Life Technologies, Gaithersburg,Md.) for the selection of neomycin-resistant clones. The resistant cellswere pooled and named C75R. These cells expressed approximately 70,000receptors/cell by Scatchard analysis.

The first 300 bp on both 5′ and 3′ ends of the cloned fragment wassequenced and compared to the reported cDNA sequence of human p75 TNF-R.The cloned sequence was a 2.3 kb fragment covering positions 58-2380 ofthe reported p75 TNF-R sequence, which encompasses the full length ofthe p75 TNF-R-coding sequence from positions 90-1475. The 2.3 kb p75TNF-R cDNA was then subcloned into the multiple cloning site of thepCDNA3 eukaryotic expression vector. The orientation of the p75 TNF-RcDNA was verified by restriction endonuclease mapping. The final 7.7 kbconstruct, pCDTR2, carried the neomycin-resistance gene for theselection of transfected cells in G418, and the expression of the p75TNF-R was driven by the cytomegalovirus promoter (FIG. 1). The pCDTR2was then transfected into monkey kidney COS-1 cells using the calciumphosphate-DNA precipitation method. The selected clone in G418 medium,termed C75R, was identified and subcultured.

¹²⁵I was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, Calif.)and the human recombinant TNF was radiolabeled using the Chloramine-Tmethod. To determine the level of p75 TNF-R expression on C75R cells,2×10⁵ cells/well were plated into a 24-well culture plate and incubatedfor 12 to 16 hours in 5% CO₂ at 37° C. They were then incubated with2-30 ng ¹²⁵I radiolabeled human recombinant TNF in the presence orabsence of 100-fold excess of unlabeled human TNF at 4° C. for 2 hours.After three washes with ice-cold PBS, cells were lysed with 0.1N NaOHand radioactivity was determined in a Pharmacia Clinigamma counter(Uppsala, Sweden). To determine the effect of TRRE on the surface levelsof p75 TNF-R, cells were incubated with or without the TRRE-containingsupernatant for 30 min at 37° C., and then the medium was aspiratedbefore incubation with radiolabeled TNF.

Soluble p75 TNF-R was generated from C75R cells by incubation withTRRE-containing supernatant. After a 30 min incubation, the supernatantwas collected and centrifugally concentrated with Centriprep-10 filter(10,000 MW cut-off membrane) (Amicon, Beverly, Mass.) and applied to 10%acrylamide SDS-PAGE. The proteins were then electrophoreticallytransferred to a polyvinylidene difluoride membrane (Immobilon)(Millipore, Bedford, Mass.). Immunostaining was performed using thebiotin-streptavidin system (Amersham, Amersham, UK) and the peroxidasesubstrate kit DAB (Vector Laboratories, Burlingame, Calif.).

The results obtained are shown in FIG. 2, C75R had a very high level ofspecific binding of radiolabeled ¹²⁵I-TNF, while parental COS-1 cellsdid not. The number of TNF-R expressed on C75R was determined to be60,000-70,000 receptors/cell by Scatchard analysis (FIG. 2, inset). Thelevel of TNF-R expression in this clone was 40 to 50 times higher thanthat of THP-1 cells. The Kd value calculated from the TNF binding assayof C75R was 5.6×10⁻¹⁰ M. This Kd value was in close agreement to thevalues previously reported for native p75 TNF-R. Thus, transfected COS-1cells expressed high levels of human p75 TNF-R in a form that appearedto be similar to native TNF-R.

In order to measure the effect of TRRE on membrane-bound TNF-R, thefollowing experiment was performed. C75R cells were seeded at a densityof 2×10⁵ cells/well in a 24-well cell culture plate and incubated for 12to 16 hours at 37° C. in 5% CO₂. The medium in the wells was aspirated,replaced with fresh medium alone or with TRRE medium, and incubated for30 min at 37° C. Throughout the examples, the “TRRE-medium” was thatcollected by stimulation of THP-1 cells with PMA followed by incubationof the cells in fresh medium for 2 hours as described. After thisincubation, the medium was replaced with fresh medium containing 30ng/ml ¹²⁵I-labeled TNF. After 2 hours at 4° C., the cells were lysedwith 0.1 N NaOH and the level of bound radioactivity was measured. Thelevel of specific binding of C75R by ¹²⁵I-TNF was significantlydecreased after incubation with TRRE. The radioactive count was 1,393cpm on the cells incubated with TRRE compared to 10,567 cpm on the cellsnot treated with TRRE, a loss of 87% of binding capacity.

In order to determine the size of the p75 TNF-R cleared from C75R byTRRE, the following experiment was performed. 15×10⁶ C75R cells wereseeded in a 150 mm cell culture plate and incubated at 37° C. in 5% CO₂for 12 to 16 hours. TRRE medium was incubated with C75R cells in the 150mm plate for 30 min and the resulting supernatant was collected andcentrifuged. The concentrated sample was applied to 10% acrylamideSDS-PAGE and electrophoretically transferred to a polyvinylidenedifluoride membrane (Immobilon). Immunostaining resulted in a singleband of 40 kDa, similar to the size found in biological fluids (FIG. 4).

The following method and assay were used throughout the Examples tomeasure TRRE activity. C75R cells and COS-1 cells were seeded into24-well culture plates at a density of 2.5×10⁵ cells/ml/well andincubated overnight (for 12 to 16 hours) in 5% CO₂ at 37° C. Afteraspirating the medium in the well, 300 μl of TRRE medium was incubatedin each well of both the C75R and COS-1 plates for 30 min in 5% CO₂ at37° C. (corresponding to A and C mentioned below, respectively).Simultaneously, C75R cells in 24-well plates were also incubated with300 μl of fresh medium or buffer (corresponding to B mentioned below).The supernatants were collected, centrifuged, and then assayed for theconcentration of soluble p75 TNF-R by ELISA as described above.

The following values were assigned and calculations made. A=(amount ofsoluble p75 TNF-R in a C75R plate treated with the TRRE containingsample); i.e. the total amount of sTNF-R in a C75R plate. B=(amount ofsoluble p75 TNF-R spontaneously released in a C75R plate treated withonly medium or buffer containing the same reagent as the correspondingsamples but without exogenous TRRE); i.e. the spontaneous release ofsTNF-R from C75R cells. C=(amount of soluble p75 TNF-R in a COS-1 platetreated with the TRRE sample or the background level of soluble p75TNF-R released by THP-1.); i.e. the degraded value of transferred(pre-existing) sTNF-R in the TRRE sample during 30 min incubation in aCOS-1 plate. This corresponds to the background level of sTNF-R degradedin a C75R plate.

The net release of soluble p75 TNF-R produced only by TRRE activityexisting in the initial sample is calculated as follows: (Net release ofsoluble p75 TNF-R only by TRRE)=A-B-C. We assigned the net release valueof soluble p75 TNF-R as the amount of TRRE activity and defined 1 pg ofsoluble p75 TNF-R net release (A-B-C) as one unit (U) of TRRE activity.

Once the TRRE assay was devised, the time course of receptor sheddingwas assayed by the following method. TRRE-medium was incubated with C75Rand COS-1 cells for varying lengths of time between 5 and 90 min. Thesupernatants were then collected and assayed for the level of solublep75 TNF-R by ELISA and the net TRRE activity was calculated as describedabove. Detectable levels of soluble receptor were released by TRREwithin 5 min and increased up to 30 min (FIG. 5A). Subsequentexperiments with longer incubation times showed that the level of TRREremained relatively constant after 30 min, presumably from the depletionof substrates (FIG. 5B). Therefore, 30 min was determined to be theoptimal incubation time for this assay system.

The binding assay clearly showed that the parental COS-1 cells did notbind human ¹²⁵I-TNF, whereas the transfected C75R cells showed strongspecific binding. Scatchard analysis indicated receptor levels of 70,000per cell which were 40 to 50 times higher than that typically found onother cell lines. This higher level of substrate allowed detection ofTRRE activity with much more sensitivity than with other cell lines. TheKd value calculated from Scatchard analysis was 5.6×10⁻¹⁰ M, similar tothe values previously reported for the native human p75 TNF-R. Thus, thetransfected cells provided the membrane form of the receptor in itsnative configuration, resulting in an excellent source of substrate.

When C75R cells were incubated with TRRE medium, soluble p75 TNF-R wasreleased into the supernatant which was measurable by ELISA. The amountof receptors released corresponded to level of TRRE activity. As C75Rcells were incubated with TRRE medium, another well of C75R cells wassimultaneously incubated with medium or buffer alone to measure thelevel of spontaneous release by C75R. The spontaneous release can be dueto an endogenous source of proteolytic enzyme, a homolog of the humanTRRE of monkey origin. In addition, TRRE medium was incubated with theparent COS-1 cells to detect the level of soluble receptors that waspre-existing in the sample. For this purpose, rather than directlymeasuring the level of soluble receptors in the supernatant by ELISA, weincubated the sample with COS-1 cells because we found that afterincubation for 30 min with COS-1 cells, significant degradation of thesoluble receptors was observed. The level of initial soluble receptorsin the supernatant may decrease up to 50% after a 30 min incubation withCOS-1 cells. Incorporating these two sources of background solublereceptors was the most accurate way to calculate the net TRRE activity.

The premise that increase in the level of soluble receptors in thesupernatant was due to the proteolytic cleavage of membrane boundreceptors was also supported by the loss of binding of ¹²⁵I-labeled TNFto C75R cells after incubation with TRRE. Since the receptor generatedby TRRE was similar in size to that found in biological fluids, thisreinforced the finding that TRRE generates sTNF-R in vivo.

EXAMPLE 3 Mechanism of TRRE Production

In Example 2, a novel assay system was used to detect and quantitate theproteolytic activity of TRRE. Using this assay system, the mechanism ofTRRE production was further investigated including (a) PMA requirement,(b) FCS dependence, (c) universality among other human cell lines andmonocytes besides THP-1, (d) time course for secretion, (e) effect ofPMA on TRRE secretion and synthesis, and (f) physiological inducersother than PMA.

THP-1 (a human monocytic leukemia cell line), U-937 (a human histiocyticlymphoma cell line), HL-60 (a human promyelocytic leukemia cell line),Raji (a human Burkitt lymphoma cell line) and K-562 (a human myelogenousleukemia cell line), which grow in suspension, and ME-180 (a humanepidermoid carcinoma cell line) and MRC-5 (a human lung fibroblast cellline), which grow adherently, were purchased from American Type CultureCollection (Rockville, Md.). These cell lines were passed twice a weekin RPMI-1640/10% heat-inactivated FCS.

Mononuclear cells were harvested at the interface of an isotonic Ficollcushion (specific gravity, 1.05) from a Leukopac obtained from normalhealthy volunteers according to the manufacturer's instructions (Sigma).Then monocytes were isolated by counterflow centrifugal elutriation witha Beckman JE-5.0 system. On average, the purity of the monocyte fractionwas over 95%, as judged by morphologic examination and nonspecificesterase staining, and viability was over 98% as assessed by the trypanblue dye exclusion test.

THP-1 cells and human peripheral blood monocytes at a density of 1×106cells/ml in RPMI-1640/1% FCS-contained were stimulated with 10-8, 10-7and 10-6 M PMA for 30 min in 5% CO2 at 37° C. The stimulated cells werewashed with serum-free medium and resuspended in the same volume ofPMA-free medium with 1% FCS for 2 more hours in 5% CO₂ at 37° C. Then,the culture supernatants were collected and assayed for TRRE activity.

THP-1 cells and human monocytes were stimulated with 10⁻⁸, 10⁻⁷ and 10⁻⁶M PMA and each supernatant was assayed for TRRE activity as described.10⁻⁶ M PMA had a consistent, strong stimulation of THP-1 cells and humanmonocytes, inducing TRRE release at concentrations of 1,304 and 883U/ml, respectively (Table 1). 10⁻⁷ M PMA stimulation induced relativelylow amounts of TRRE and 10⁻⁸ M PMA stimulation did not induce any TRREfrom either monocytes or THP-1 cells (data not shown). PMA stimulationat the concentration of 10⁻⁶ M was adopted in all subsequent experimentsfor the induction of TRRE from THP-1 cells.

TABLE 1 Cell Activator TRRE Activity (U) Human Monocyte PMA 10⁻⁷ M 5110⁻⁶ M 883 THP-1 PMA 10⁻⁷ M 368 10⁻⁶ M 1,304

Suspension cell lines including THP-1, U-937, HL-60, Raji and K-562 at adensity of 1×10⁶ cells/ml were stimulated in RPMI-1640/1% FCS-with 10⁻⁶M PMA for 30 min in 5% CO₂ at 37° C. After pelleting the cells anddiscarding the supernatants, the cells were washed once with serum-freemedium and then incubated for 2 more hours at the same density of 1×10⁶cells/ml in PMA-free RPMI-1640/1% FCS. Adherent cell lines includingME-180 and MRC-5 were seeded in 100 mm cell culture plates withRPMI-1640/10% FCS-at a density of 6×10⁶ cells/10 ml/plate in 5% CO₂ at37° C. overnight. After discarding the medium from the 100 mm plates,these adherent cells were stimulated in 6 ml of RPMI-1640/1% FCS with10⁻⁶ M PMA (at an approximate density of 1×10⁶ cells/ml) for 30 min in5% CO₂ at 37° C. Following washing of the plates with serum-free medium3 times, the cells were incubated for 2 more hours in 6 ml of PMA-freeRPMI-1640/1% FCS. These supernatants from suspension and adherent cellswere collected and assayed for TRRE activity (as described in Example1).

10⁻⁶ M PMA-stimulated suspension cell lines including THP-1, U-937,HL-60, Raji, and K-562 and adherent cell lines including ME-180 andMRC-5 produced TRRE activity at concentrations of 2,884 U/ml, 3,288U/ml, 3,144 U/ml, 2,390 U/ml, 3,356 U/ml, 1,694 U/ml, and 1,477 U/ml per1×10⁶ cells, respectively (Table 2). Thus, TRRE can be induced not onlyby THP-1 cells but also by all cell lines investigated withPMA-stimulation.

TABLE 2 TRRE Cell line (U/ml/10⁶ cells) THP-l (human monocytic leukemia)2,884 U-937 (human histiocytic lymphoma) 3,288 HL-60 (humanpromyelocytic leukemia) 3,144 ME-180 (human epidermoid carcinoma) 1,694MRC-5 (human lung fibroblast) 1,477 Raji (Burkitt lymphoma) 2,390 K-562(human myelogenous leukemia) 3,356

THP-1 cells were stimulated with 10−6 M PMA in RPMI-1640 with 1% FCS for30 min. The stimulated cells were then washed and incubated for 2 morehours in PMA-free RPMI 1640 with 0%, 1%, and 10% FCS, which led to therelease of 224, 1,356, and 2,275 U/ml TRRE, respectively (Table 3). Thissuggests that some serum factors are required by cells for a normalresponse to PMA.

TABLE 3 FCS Concentration TRRE Activity (U/ml)  0% 224  1% 1,356 10%2,275

THP-1 cells were stimulated with 10⁻⁶ M PMA in RPMI-1640 with 1% FCS for30 min and then washed and resuspended in the same volume of PMA-freemedium with 1% FCS. The cells were incubated for additional 2 to 23hours making the total induction time of TRRE 3 to 24 hours from initialstimulation. This kinetic study revealed that the release of TRRE intoculture supernatants peaked as early as 3 hours, followed by a gradualdecline afterwards, while the level of sTNF-R derived from THP-1 cellsincreased over time (FIG. 6). Consequently, in order to obtain higherTRRE activity with lower sTNF-R background, 2 hours incubation (total 3hours induction) was adopted in subsequent experiments.

TRRE media were serially diluted up to 1:256 dilution. Detectable levelsof TRRE activity were present in samples diluted 16-fold (FIG. 7). Whilethe level of sTNF-R present in TRRE supernatants decreased in proportionto their dilution, no significant differences in TRRE activity werefound between the original and 2-fold diluted supernatant, suggesting adepletion of substrates and that the level of TRRE activity in theoriginal supernatant might be saturating the assay system.

Several inhibitors including cycloheximide (Chx) (an inhibitor ofprotein synthesis), actinomycin D (ActD) (an inhibitor of RNAsynthesis), N-ethylmaleimide (NEM) (an inhibitor of membraneinternalization and movement), cytochalasin B (CytB) (an inhibitor ofmicrofilament formation), and colchicine (Col) (an inhibitor ofmicrotubule formation) were purchased from Sigma Chemical (St. Louis,Mo.). THP-1 cells at a density of 1×10⁶ cells/ml in RPMI-1640/1% FCSwith 10⁻⁶ M PMA was co-stimulated along with 10 μg/ml Chx, 1 μg/ml ActD,1 mM NEM, 0.1 mM CytB, or 0.1 mM Col for 30 min at 37° C. in 5% CO₂.Following centrifugation at 400×g for 5 min, the supernatant wasdiscarded and the cells were washed once with serum-free medium. Thecells were incubated for an additional 2 hours at the same density(1×10⁶ cells/ml) in PMA-free RPMI-1640 with corresponding inhibitors.These supernatants were collected and assayed for TRRE activity. Thesupernatant from THP-1 cells stimulated only with 10⁻⁶ M PMA, butwithout any inhibitor, was used as a control. % TRRE production wasexpressed by dividing TRRE activity induced with PMA plus inhibitors bythat of the control.

To understand the mechanism of PMA in the production of TRRE, severalinhibitors were co-incubated with PMA as described. 10 μg/ml Chx, 1μg/ml ActD, 1 mM NEM, 0.1 mM CytB, and 0.1 mM Col modified PMA-inducedTRRE activity to 104%, 97%, 17%, 91%, and 111%, respectively (Table 4).The results obtained indicate that only NEM inhibited PMA-induced TRREproduction, suggesting that membrane internalization and movement areonly involved in the release of TRRE induced by PMA. Protein synthesis,RNA synthesis and transmission within the cytoplasm may not be requiredfor PMA-induced TRRE release.

TABLE 4 % of TRRE Inhibitor Concentration production Actinomycin D 1μg/ml 104 Cycloheximide 10 μg/ml 97 N-ethyl-maleimide 1 mM 17Cytochalasin B 0.1 mM 91 0.01 mM 103 Colchicine 1 mM 125 0.1 mM 111 0.01mM 95

THP-1 cells at a density of 1×10⁶ cells/ml were stimulated in 1%FCS-contained RPMI-1640 with 10⁻⁶ M PMA for 30 min at 37° C. in 5% CO₂.After washing the cells with serum-free medium to remove PMA, the cellswere incubated in the same volume of 1% FCS-contained RPMI-1640 withoutPMA for 2 hours at 37° C. in 5% CO₂. Then the cells were centrifugeddown and the supernatant was collected for TRRE assay. These pelletedcells were again resuspended in 1% FCS-contained RPMI-1640 with orwithout 10⁻⁶ M PMA and incubated for 30 min at 37° C. in 5% CO₂. Afterwashing the cells with serum-free medium to remove PMA, the cells,stimulated or non-stimulated by PMA, were incubated in the same volumeof 1% FCS-contained RPMI-1640 without PMA for 2 and 4 hours each at 37°C. in 5% CO₂. Then, all 4 types of supernatants were collected for TRREassay (as described in Example 1).

THP-1 cells were stimulated with PMA twice to investigate the successiveinduction of TRRE as described. By the first stimulation of PMA, THP-1cells released 951 U/ml TRRE. Then, these cells, which were againstimulated by PMA for 30 min and incubated for additional 2 hours and 4hours in PMA-free medium, released 1,245 U/ml and 1,044 U/ml TRRE,respectively (FIG. 8). On the other hand, after the first stimulation ofPMA, the THP-1 cells, which were again incubated for 2 hours and 4 hoursin PMA-free medium following 30 min incubation in PMA-free medium andwashing, released no TRRE activity. These data revealed that TRRE couldbe released again by a second PMA-stimulation with nearly identicalconcentrations between 2 hours and 4 hours incubation; whereas new TRRErelease was not detected without the second PMA-stimulation even thoughonce stimulated with PMA. According to these data, the response of THP-1cells to PMA for the release of TRRE may be very quick and repeatable,however the response does not last more than 2 hours once PMA isremoved.

THP-1 cells at a density of 1×6 cells/ml were stimulated for 2 hours in1% FCS-contained RPMI-1640 alone, or with cytokines including IL-1β (10ng/ml), IL-2 (4 μg/ml), IL-4 (10 ng/ml), IL-6 (100 ng/ml), IL-10 (100ng/ml), TNF (1 μg/ml), LT (1 μg/ml) and IFN-γ (100 ng/ml), and hormonesincluding epinephrine (10⁻⁶ M), insulin (10⁻⁷ M), prostaglandin E₂(PGE₂) (10⁻⁷ M) and hydrocortisone (10⁻⁶ M). After pelleting the cells,culture supernatants were collected and assayed for TRRE activity. Forthe TRRE assay, the level of spontaneous release of soluble p75 TNF-Rwas obtained by incubating C75R cells in RPMI-1640/1% FCS whichcontained only the stimulants described above, and no exogenous TRRE.

We have shown that PMA is a very strong and rapid inducer of sTNF-R.Gatanaga et al (1991); and Hwang et al. (1993) J. Immunol.151:5631-5638. However, in order to determine physiological inducers ofTRRE other than PMA, several cytokines and hormones were investigated asdescribed. Among cytokines including IL-1β, IL-2, IL4, IL-6, IL-10, TNF,LT, and IFN-γ, and hormones including epinephrine, insulin, PGE₂, andhydrocortisone, epinephrine and IL-10 induced TRRE activity with 2 hoursstimulation. The level of TRRE activity induced by IL-10 andepinephrine, was significantly lower than that caused by PMA.

Tables 1 and 2 showed that the TRRE induction protocol and its assaysystem were not only effective to all the tumor cell lines investigated,but also to normal human monocytes. Therefore, TRRE activity is probablynot a unique characteristic of transformed cell lines, but a commontrait possessed by most human cells. This is in agreement with theprevious work that reported most human cells express both TNF-Rssimultaneously. Gehr et al. (1992); Naume et al. (1991) J. Immunol.146:3045-3048; and Porteu et al. (1991) J. Biol. Chem. 266:18846-18853.Thus, cells can autoregulate their susceptibility to TNF by controllingthe number of their own TNF-R by synthesizing TRRE. The reason that aPMA-concentration as high as 10⁻⁶ M was required to observe an effectiveinduction of TRRE can be due to the protocol used. Here, THP-1 cellswere pulse stimulated for 30 min, washed once to remove PMA andincubated in PMA-free medium for 2 more hours for the induction of TRRE.Also, it is quite possible that these cells release high levels ofactive TRRE immediately when stimulated with PMA during the 30 min pulsestimulation. The TRRE released during this period can be discarded whenthe cells are washed and incubated with fresh medium.

Although the production of TRRE was FCS-dependent, FCS-enriched mediumalone in the absence of PMA-stimulation did not induce TRRE activity.Therefore, some serum factors may assist in the secretion of TRREinduced by PMA, while serum itself does not induce. Since the incubationof PMA-stimulated THP-1 cells in the presence of 1% FCS wouldsignificantly decrease the level of contaminating proteins from FCS andincrease the specific activity of TRRE (the value of TRRE units/A280) inthe supernatant, TRRE induction at 1% FCS concentration was adopted forsubsequent experiments.

PMA is an extremely strong and rapid inducer of TRRE and, indirectly,TNF-R. After a 30 min stimulation of THP-1 cells with 10⁻⁶ M PMA thesecretion of TRRE started immediately and reached its maximum as quicklyas within 2 hours. This suggests that TRRE is already stored in THP-1cells ready to be secreted in response to PMA-stimulation. Basically,PMA is a powerful stimulator of protein kinase C which is anchoredinside the cell membrane once activated. Thus, it is likely that (i)TRRE is stored in the cytoplasm very close to the cell membrane ready tobe secreted through the protein kinase C cascade by PMA stimulation;(ii) TRRE is a peripheral (or extrinsic) membrane protein which isdissociated from the membrane through the change of interactions withother proteins or with any phospholipid by stimulated protein kinase C;or (iii) TRRE is an integral (or intrinsic) membrane protein which iscleaved and secreted to be an active form after its cytoplasmic portioninteracts directly or indirectly with protein kinase C.

TRRE induction by PMA did not require de novo protein synthesis, RNAsynthesis and transmission inside the cytoplasm, but only membraneinternalization and movement. This is compatible with the data that TRREreleased very quickly by PMA-stimulation and halted once PMA wasremoved. With PMA-stimulation, however, TRRE synthesis began at the sametime as TRRE release. After the initial release, TRRE accumulated insidethe cell or on the cell surface within 2 hours ready to be secreted bythe next stimulation. Evidence for direct cleavage of TNF-R is that theshedding of sTNF-R occurs very quickly (5 min) and with maximal sheddingwithin 30 min.

Except for PMA, shedding of sTNF-R has been known to be enhanced byseveral cytokines including TNF, IL-1, IL-6, IL-10 and IFN, leukocytemigration enhancement factors includingformyl-methionyl-leucyl-phenylalanine (fMLP) and C5a, and calciumionophore. Gatanaga (1993) Lymphokine Res. 12:249-253; Porteu (1994) J.Biol. Chem. 269:2834-2840; van der Poll (1995) J. Immunol.155:5397-5401; Porteu et al. (1991); and Porteu and Natah (1990) J. Exp.Med. 172:599-607. In the experiments provided herein, IL-10 andepinephrine induced TRRE from THP-1 cells with 2 hour stimulation,though their induction was not as strong as PMA.

IL-10 is a potent inhibitor of monocyte- and macrophage-functions. Moore(1993) Annu. Rev. Immunol. 11:165-190. IL-10 has anti-inflammatoryactivity on monocytes and inhibits the release of pro-inflammatorycytokines including TNF and IL-1. Bogdan et al. (1991) J. Exp. Med.174:1549-1555; Fiorentino et al. (1991) J. Immunol. 147:3815-3822; deWaal Malefyt et al. (1991) J. Exp. Med. 174:1209-1220; Katsikis et al.(1994) J. Exp. Med. 179:1517-1527; Joyce et al. (1994) Eur. J. Immunol.24:2699-2705; and Simon et al. (1994) Proc. Natl. Acad. Sci. USA91:8562-8566. Elevated levels of IL-10 have been detected in plasma ofpatients with sepsis, and after administration of LPS to animals.Marchant et al. (1994) Lancet 343:707-708; Derkx et al. (1995) J.Infect. Dis. 171:229-232; Durez et al. (1993) J. Exp. Med. 177:551-555;and Marchant et al. (1994) Eur. J. Immunol. 24:1167-1171. In vivo, IL-10has also been shown to protect mice against endotoxin shock. Gerard etal. (1993) J. Exp. Med. 177:547-550; and Howard et al. (1993) J. Exp.Med. 177:1205-1208. IL-10 leads to increased levels of mRNA for p75TNF-R, increased release of soluble p75 TNF-R and a concomitantreduction of surface expression of p75 TNF-R on monocytes. Joyce et al.(1994). Thus, IL-10 may be considered to reduce the pro-inflammatorypotential of TNF by (i) inhibiting the release of TNF itself, and (ii)down-regulating surface TNF-R expression while (iii) increasingproduction of sTNF-R capable of neutralizing TNF cytotoxicity. Joyce etal. (1994); and Leeuwenberg et al. (1994) J. Immunol. 152:4036-4043. Thedata presented herein that IL-10 may induce TRRE activity are consistentwith these findings and indicate a newly revealed function of IL-10 asan anti-inflammatory cytokine.

In stressful situations including endotoxic shock, serum levels ofcatecholamines and glucocorticoids are elevated chiefly from adrenalmedulla and adrenal cortex, respectively, in response to high serumlevel of adrenocorticotropic hormone (ACTH) throughout the whole bodysystem. TNF also has been implicated in the early metabolic eventsfollowing stressful situations, and infusion of recombinant TNF in dogswas associated with increase of serum levels of catecholamines,glucocorticoids and glucagon. Tracey et al. (1987) Surg Gynecol. Obstet.164:415-422. On the other hand, as a local phenomenon, epinephrine andnorepinephrine are found in macrophages which express β-adrenergicreceptors and these endogenous catecholamines seem to regulateLPS-induced TNF production in an autocrine fashion in vitro. Hjemdahl etal. (1990) Br. J. Clin. Pharmacol. 30:673-682; Hjemdahl et al. (1990)Br. J. Clin. Pharmacol. 30:673-682; Talmadge et al. (1993) Int. J.Immunopharmacol. 15:219-228; and Spengler et al. (1994) J. Immunol.152:3024-3031. Actually, exogenous epinephrine and isoproterenol, aspecific β-adrenergic agonist, inhibit the production of TNF from humanblood and THP-1 cells stimulated by LPS. Hu et al. (1991) J.Neuroimmunol. 31:35-42; and Severn (1992) J. Immunol. 148:3441-3445.

While epinephrine may be an important endogenous inhibitor of TNFproduction, especially in sepsis, epinephrine also decreases the numberof TNF-R on macrophages. Bermudez et al. (1990) Lymphokine Res.9:137-145. We have shown that in trauma patients both p55 and p75 TNF-Rlevels were significantly elevated within 1 hour of injury along withhigh serum level of epinephrine. Tan et al. (1993) J. Trauma 34:634-638.These findings are in agreement with the data that epinephrine inducedTRRE activity and may lead to the increase of sTNF-R.

Insulin and glucagon have the function to down-regulate TNF-R inaddition to epinephrine. Bermudez et al. (1990). Many inflammatorycytokines besides IL-10 may influence the shedding of sTNF-R includingTNF, IL-1, IL-6, and IFN for up-regulation and IL-4 for down-regulation.van der Poll et al. (1995); Gatanaga et al. (1993); and Joyce et al.(1994). According to the data presented herein, insulin, IL-1β and IL-6induced sTNF-R from THP-1 cells with 2 hours stimulation but no apparentTRRE activity, suggesting that the sTNF-R may be produced by (a)protease(s) other than TRRE or by another form of TRRE which may bemembrane-bound. Thus, there may be at least two pathways responsible forthe shedding of sTNF-R, not only in vitro but also in vivo. In vivo, oneis seen in trauma patients, who experience a rapid increase of sTNF-R inthe serum. This pattern of increase is similar to that caused by PMAstimulation and therefore is presumably mediated by TRRE. Anotherpathway in vivo is involved in chronic or spontaneous induction ofsTNF-R seen in cancer patients and even in healthy individuals. Gatanagaet al. (1990a); and Gatanaga et al. (1990b). Presumably (a) protease(s)other than TRRE or various forms of TRRE including a membrane-bound formactivated by cleavage to a soluble form could be responsible for thisincrease of sTNF-R. However, the induction of sTNF-R in cancer patientsmay be at least partially due to increased TRRE activity. This activitywas generally higher in human lung tumors than in non-canceroussurrounding tissues. As shown in FIG. 20, in 9 out of 12 cases, TRREactivity was higher in culture supernatants of tumor cells than in thatof adjacent non-tumor cells. In cases 2, 3, 5, 9, and 11, the TRREactivity was approximately 75%, 50%, 30%, 36%, and 60% higher,respectively, in tumor than in non-tumor cells.

EXAMPLE 4 Physiologic Properties of TRRE

In this Example, the physiologic properties of TRRE were investigated,including (a) stability versus temperature and pH, (b) metalrequirements, (c) mechanistic class of protease, (d) comparison withknown proteases, especially MMPs, and (e) conformational relationshipbetween sTNF-R and TRRE. The example further provides partialpurification steps and results for TRRE.

In order to purify TRRE, TRRE medium was concentrated by 100% saturatedammonium sulfate precipitation method at 4° C. The precipitate waspelleted by centrifugation at 10,000×g for 30 min and resuspended in PBSin approximately twice the volume of the pellet. This solution was thendialyzed at 4° C. against 10 mM Tris-HCl, 60 mM NaCl, pH 7.0. Thissample was loaded on an anion-exchange chromatography, Diethylaminoethyl(DEAE)-Sephadex A-25 column (Pharmacia Biotech) (2.5×10 cm) previouslyequilibrated with 50 mM Tris-HCl, 60 mM NaCl, pH 8.0. TRRE was theneluted with an ionic strength linear gradient of 60 to 250 mM NaCl, 50mM Tris-HCl, pH 8.0. Each fraction was measured for absorbance at 280 nmand assayed for TRRE activity. The DEAE fraction with the highestspecific activity (the highest value of TRRE units/A280) was pooled andused in the characterizations of TRRE described in this example. Thesesamples are termed herein “partially-purified” TRRE.

The partially-purified TRRE was preincubated at 37° C. for 30 min withseveral classes of protease inhibitors. Protease inhibitors werepurchased from Sigma Chemical (St. Louis, Mo.), includingphenylmethylsulfonyl fluoride (PMSF), 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF), 3,4-dichloroisocoumarin (3,4-DCI), N-α-tosyl-L-lysinechloromethyl ketone (TLCK), N-tosyl-L-phenylalanine chloromethyl ketone(TPCK), ethylenediaminetetraacetic acid (EDTA), ethyleneglycol bis(2-aminoethyl ether) tetraacetic acid (EGTA), 1,10-phenanthroline, andphosphoramidon. These inhibitors can be divided into the followingclasses: (i) serine protease inhibitors (4 mM PMSF, 0.5 mM AEBSF, and0.1 mM 3,4-DCI); (ii) serine and cysteine protease inhibitors (0.1 mMTLCK and TPCK); (iii) chelating agents (2 mM EDTA, EGTA, and1,10-phenanthroline); (iv) a metallo-endoprotease inhibitor (0.5 mMphosphoramidon); and (v) divalent heavy metal ions (2 mM CaCl₂, MgCl₂,MnCl₂, ZnCl₂, CoCl₂, CuCl₂, and FeCl₂). After the preincubation, thesamples were assayed for TRRE activity. For the TRRE assay, spontaneousrelease of soluble p75 TNF-R in each sample from C75R cells was obtainedby incubating C75R cells with the corresponding reagents without TRRE.Partially-purified TRRE preincubated at 37° C. for 30 min without anyreagent and assayed for TRRE activity was taken as a control. Thepercent activity remaining (% control) was expressed relative to thecontrol.

The partially-purified TRRE was preincubated with 4 mM CaCl₂, 0.1 mMZnCl₂, 2 mM 1,10-phenanthroline, 2 mM 1,10-phenanthroline plus 2 mMCaCl2, or 2 mM 1,10-phenanthroline plus 2 mM ZnCl2 at 37oC. for 30 minprior to assaying for TRRE activity. The percent activity remaining (%control) was calculated as above.

The results obtained are depicted in Table 6. Partial inhibition of TRREactivity was obtained by chelating agents such as 1,10-phenanthroline,EDTA and EGTA (% TRRE activities remaining were 41%, 67% and 73%,respectively, at 2 mM concentration), which are potent inhibitors ofmetalloproteases (Table 5). On the other hand, serine proteaseinhibitors such as PMSF, AEBSF and 3,4-DCI, and serine and cysteineprotease inhibitors such as TLCK and TPCK had no effect on theinhibition of TRRE. These data suggest that TRRE requires (a) metalion(s) for its activity. To assess further the metal requirement ofTRRE, the enzymatic activity was assayed in the presence of divalentmetal ions at 2 mM concentration. TRRE was slightly activated in thepresence of Mn²⁺, Ca²⁺, Mg²⁺, and Co²⁺ (% TRRE activities remaining were157%, 151%, 127%, and 123%, respectively), whereas partial inhibitionoccurred in the presence of Zn²⁺ and Cu²⁺ (% TRRE activities remainingwere 23% and 47%, respectively) (Table 5).

TABLE 5 Concentration % Control of Inhibitor (mM) TRRE activity PMSF 4116 ± 4  AEBSF 0.5 92 ± 8  TLCK 0.1 108 ± 5  TPCK 0.1 107 ± 7  3,4-DCI0.1 108 ± 4  EDTA 2 67 ± 7  EGTA 2 73 ± 4  1,10-Phenanthroline 2 41 ± 6 Phosphoramidon 0.5 84 ± 13 Ca²⁺ 2 151 ± 23  Mg²⁺ 2 127 ± 9  Mn²⁺ 2 157 ±33  Zn²⁺ 2 23 ± 15 Co²⁺ 2 123 ± 15  Cu²⁺ 2 47 ± 21 Fe²⁺ 2 98 ± 8 

As shown in Table 6, TRRE with 4 mM Ca²⁺ and 0.1 mM Zn²⁺ showed 189±4%and 122±6% activity (% TRRE activities remaining), respectively (Table5). As shown previously in Table 7, however, high concentrations of Zn²⁺(over 2 mM) partially inhibited TRRE activity. Therefore, Zn²⁺ has twoopposite effects on TRRE activity based upon its concentrations, whileCa²⁺ can activate TRRE at any concentration. TRRE with 2 mM1,10-phenanthroline, which is a potent inhibitor of metalloproteases,partially inhibited TRRE and the addition of 2 mM Ca²⁺ or 2 mM Zn²⁺partially restored TRRE activity inhibited by 2 mM 1,10-phenanthroline.Thus, at least Ca²⁺ and Zn²⁺ modulated TRRE activity. Since the activityof TRRE appears to be metal dependent, TRRE can be ametalloprotease-like enzyme.

TABLE 6 % Control of TRRE Activity Ca²⁺ (4 mM) 189 ± 4  Zn²⁺ (0.1 mM)  122 ± 6  1,10-Phenanthroline (2 mM) 47 ± 9 + Ca²⁺ (2 mM)  80 ± 15 + Zn²⁺(2 mM) 62 ± 6

In order to detect MMP activities, aliquots of crude andpartially-purified TRRE samples were assayed by gelatin, casein,elastin, and type I collagen zymography according to the methoddescribed by Hibbs et al. (1985) J. Biol. Chem. 260:2493-2500 withslight modifications. Briefly, these samples were dissolved innonreducing Laemmli sample buffer without boiling and separated byelectrophoresis using an 8% SDS polyacrylamide slab gel impregnated with1 mg/ml gelatin, casein, elastin, and type I collagen. Afterelectrophoresis, the gel was washed twice, to remove SDS, with 50 mMTris-HCl buffer, pH 7.6, containing 5 mM CaCl₂, 1 μM ZnCl₂, 2.5% TritonX-100 (v/v) for 30 min at room temperature with shaking, followed by abrief rinse in washing buffer without Triton X-100. The gel was thenincubated in 50 mM Tris-HCl buffer, pH 7.6, containing 5 mM CaCl₂, 1 μMZnCl₂, 1% Triton X-100, 0.02% NaN₃ at 37° C. overnight with shaking. Theenzymatic reaction was terminated by 10% acetic acid, followed bystaining with 0.1% Coomassie Brilliant Blue R-250 and destaining with asolution of 10% acetic acid and 10% methanol.

In this assay, clear zones against the blue background indicate thepresence of gelatinolytic, caseinolytic, elastinolytic, and type Icollagenolytic activity in gelatin, casein, elastin, and type I collagenzymography, respectively. These MMP activities on zymography gels werecompared with the TRRE activity of the corresponding samples. For the 4crude TRRE samples, THP-1 cells at a density of 1×10⁶ cells/ml werestimulated with or without 10⁻⁶ M PMA for 30 min in 0% or 1%FCS-containing RPMI-1640 followed by 2 more hours incubation in PMA-freemedium with the same concentrations of FCS. The partially-purified TRREsample was prepared from serum-free TRRE source as described. For thepositive control of MMPs, THP-1 cells were incubated in serum-freeRPMI-1640 with 10⁻⁸ M PMA for 24 hours, and after the culture medium waswashed away, the cells were incubated in fresh serum-free medium withoutPMA for additional 24 hours and then the supernatant was harvested.

Previously, MMPs have been reported to be responsible for the cleavageof pro-TNF. Gearing et al. (1994); and Gearing et al. (1995). Since TRREappears to be a metalloprotease, zymography was performed to detect MMPactivities in TRRE samples. Zymography on gelatin-, casein-, elastin-,and type I collagen-containing gels may primarily detect 72 kDgelatinase A and 92 kD gelatinase B (MMP-2 and -9), stromelysin (MMP-3)and matrilysin (MMP-7), macrophage metalloelastase (MMP-12), andinterstitial collagenase (MMP-1), respectively. These MMPs have beenshown to be secreted by macrophages. Hibbs et al. (1985); Chin et al.(1985) J. Biol. Chem. 260:12367-12376; Miyazaki et al. (1990) CancerRes. 50:7758-7764; Senior et al. (1991) J. Biol. Chem. 266:7870-7875;Dansette et al. (1979) Anal. Biochem. 97:340-345. FIGS. 10A and 10B showthe relationship between TRRE activity and gelatin zymography on crudeand partially-purified TRRE samples, respectively. Each is arepresentative example of four different substrate-impregnatedzymography gels. Only PMA-stimulated THP-1 cells in 0% and 1%FCS-contained supernatants produced TRRE at concentrations of 217 and2,096 U/ml, respectively (FIG. 10A). The latter crude sample with 1% FCSwas treated in the same manner as the enzyme source of TRRE previouslydescribed. On gelatin zymography, two gelatinolytic bands derived fromgelatinase A and B were detected only in three 1% FCS-contained sampleswith completely equal intensity, suggesting that these MMP activitiesresulted not from TRRE but from 1% FCS. No gelatinolytic activity wasdetected in partially-purified TRRE without FCS, contrary to its highTRRE activity (3,514 U/ml) (FIG. 10B). Despite the strong gelatinolyticactivities, the positive control had no TRRE activity. The casein,elastin; and type I collagen zymography gels showed no MMP activity ineither crude or partially-purified samples. Therefore, TRRE appears tohave a distinctly different activity than that of knownmacrophage-associated MMPs.

In order to determine the molecular weight of TRRE by gel filtration,TRRE was obtained from PMA-stimulated THP-1 cells in RPMI-1640 with 1%FCS. This source was adjusted to 100% saturation with ammonium sulfateand the precipitate was pelleted and resuspended in PBS followed bydialysis as described for the partial purification. This concentratedTRRE (1 ml) was loaded onto a Sephadex G-150 column (1.0×30 cm)(Pharmacia Biotech) which was equilibrated in 10 mM Tris-HCl, 60 mMNaCl, pH 7.0. The flow rate was 1 ml/min, and 1-ml fractions werecollected for the measurement of TRRE activity, soluble p75 TNF-R, andabsorbance at 280 nm.

The results obtained indicate that TRRE activity was detected as asingle peak accompanied by a similar profile of soluble p75 TNF-R in gelfiltration (FIG. 11). The peak elution of both TRRE and soluble p75TNF-R was at Fraction Number 10, which had migrated approximately at 150kDa molecular weight according to the standards. The same sample wasalso applied to DEAE-Sephadex chromatography and a similar profilebetween TRRE and soluble p75 TNF-R was obtained. This evidence suggeststhat some of TRRE and sTNF-R, an enzymatic product of TRRE, remain boundin the reacting solution and migrate as a complex in both gel filtrationand DEAE columns.

In order to assure that TRRE was being specifically purified, purifiedTRRE was subject to affinity chromatography on soluble p75 TNF-RSepharose affinity chromatography. The activity of partially-purifiedTRRE was adjusted to 5,000 U/ml with 10 mM Tris-HCl, 60 mM NaCl, pH 7.0.This diluted TRRE was incubated with C75R cells growing in logarithmicphase in a 150 mm cell culture plate for 30 min at 37° C. in 5% CO₂ (15ml of TRRE sample per plate). The supernatants from five plates (75 ml)were collected and centrifugally concentrated to approximately 2 ml withCentriprep-10 filter (Amicon). This concentrated sample was applieddirectly to soluble p75 TNF-R affinity chromatography (soluble p75TNF-R- and Affigel 10 (Bio-Rad)-conjugated column) (column size; 1×2 cm)at 4° C. which was equilibrated with 10 mM Tris-HCl, 60 mM NaCl, pH 7.0.The column was then washed with 10 ml of the same Tris-buffer and elutedwith 5 ml of elution Buffer (ImmunoPure elution Buffer, Pierce). Theeluate from the affinity column was applied to gel filtration (Naps 5column, Pharmacia) to change the elution buffer for the buffer of 10 mMTris-HCl, 60 mM NaCl, pH 7.0 and 2 ml fractions were collected. Eachfraction was measured for TRRE activity and absorbance at 280 nm. Thetotal TRRE activity from the active fractions of gel filtration wasconsidered as the TRRE activity in the eluate of soluble p75TNF-R-affinity chromatography. The flow through and washing of theaffinity column, whose buffer was 10 mM Tris-HCl, 60 mM NaCl, pH 7.0,were directly measured for TRRE activity without gel filtration.

To verify the hypothesis that a bound form of TRRE and its enzymaticproduct, sTNF-R, exists in vitro, the reacting solution between TRRE andits substrate-expressing C75R cells was applied to soluble p75TNF-R-affinity chromatography as described. Among the total amount ofrecovered TRRE from soluble p75 TNF-R-affinity column (100%), 53%, 22%,and 25% was distributed in the flow through, the wash, and the elution,respectively (FIG. 12A). This data means that 25% of active TRRE maycombine with soluble p75 TNF-R affinity column even after TRRE was oncetreated with its substrate and then that their binding form might existin vivo.

The stability of partially-purified TRRE was investigated againstvarious temperatures and pH values. TRRE activity was stable when storedat −70° C. TRRE activity, however, was reduced by incubation at 4° C.for 2 days, heating at 56° C. for 30 min, and boiling for 15 min to 82%,84% and 16% of its initial activity, respectively. TRRE samples treatedat various pH levels were pre-incubated at 37° C. for 30 min and thenapplied to the TRRE assay after adjusting the pH of all samples to 7.4.TRRE samples pre-incubated at pH 6.0, 7.0, 8.0, and 9.0 showed 52%,100%, 69%, and 73%, respectively of TRRE activity contained in theoriginal supernatant (pH 7.4), respectively (Table 8). Thus, the optimalpH of TRRE was around 7.0 and its activity deteriorated more in acidicthan basic conditions.

TABLE 8 % Control of TRRE % Control of TRRE Temperature Activity pHActivity  4° C. for 48 hours 82 6.0 52  56° C. for 30 min. 84 7.0 100100° C. for 15 min. 16 8.0 69 9.0 73

Since TRRE and sTNF-R may remain associated in the reaction solution invitro, their affinity was investigated to determine whether this complexfunctioned as an inhibitor or a protector of TRRE in the enzymaticreaction. Partially-purified TRRE was incubated with C75R cells growingin logarithmic phase in a 150 mm cell culture plate for 30 min at 37° C.in 5% CO₂. TRRE activity was assayed before and after the treatment ofthe culture plate. During the incubation, the substrate (TNF-R) wasconsidered to be much more abundant than the enzyme (TRRE). Relative tothe TRRE activity before the reaction, 86% TRRE activity was detectedeven after the reaction (FIG. 12B). Therefore, although TRRE was treatedonce with excessive substrate, thereby creating conditions in whichthere is a high concentration of TRRE/sTNF-R complex, TRRE activityremained comparatively high against the next reaction. Thus, thisTRRE/sTNF-R complex form was not inhibitory for TRRE.

Due to the metal requirement of TRRE indicated by the effect ofchelating agents and divalent heavy metal ions, TRRE appears to be ametalloprotease. The reason for incomplete inhibition by chelatingagents may be because this partially purified TRRE from DEAE-Sephadexchromatography consists of several other enzymes or factors which alsohave an influence on the cleavage of TNF-R. Another explanation is thatthe concentration of chelating agents and divalent metal ions necessaryto achieve complete inhibition was not attained due to their toxicitiesto C75R cells in this assay system. The inhibition of TRRE activity by1,10-phenanthroline was partially restored by Ca²⁺ and Zn²⁺independently, suggesting that several metal ions including Ca²⁺ andZn²⁺ are related to the activity or stability of TRRE. Two reportsdescribe the involvement of a metalloprotease in the production ofsTNF-R by utilizing a specific metalloprotease inhibitor, TNF-α proteaseinhibitor (TAPI).

TAPI blocks the shedding of soluble p75 and p55 TNF-R, respectively.Crowe et al. (1995); and Mullberg et al. (1995). Moreover, theprocessing of pro-TNF on the cell membrane was reported to be dependenton a MMP-like enzyme). Gearing et al. (1994); and Gearing et al. (1995).MMPs are a family of structurally related matrix-degrading enzymes thatplay a major role in tissue remodeling and repair associated withdevelopment and inflammation. Matrisian (1990) Trends Genet. 6:121-125;Woessner (1991) FASEB J. 5:2145-2154; and Birkedal-Hansen et al. (1993)Crit. Rev. Oral Biol. Med. 4:197-250. Pathological expression of MMPs isresponsible for tumor invasiveness, osteoarthritis, atherosclerosis, andpulmonary emphysema. Mignatti et al. (1986) Cell 47:487-498; Khokha(1989) Science 243:947-950; Dean et al. (1989) J. Clin. Invest.84:678-685; Henney et al. (1991) Proc. Natl. Acad. Sci. USA88:8154-8158; and Senior et al. (1989) Am. Rev. Respir. Dis.139:1251-1256. MMPs are Zn²⁺-dependent enzymes which have Zn²⁺ in theircatalytic domains. Ca²⁺ stabilizes their tertiary structuresignificantly. Lowry et al. (1992) Proteins 12:42-48; and Lovejoy et al.(1994) Science 263:375-377. Thus, according to the similar metaldependency, TRRE may be a part of the MMPs family of which 11 MMPs havebeen cloned.

It has been reported that not only metalloproteases but also serineproteases are involved in the cleavage of TNF-R by adding PMA and serineprotease inhibitors simultaneously in the culture medium of THP-1 cells.These results indicate that at least two different kinds of proteasesare involved in the induction phase of TRRE and that these enzymes forma cascade for their activation. According to the results presentedherein, however, serine protease inhibitors had no effect towardpartially-purified TRRE samples whose enzymatic activity was alreadyestablished. Bjornberg et al. (1995); and Hwang et al. (1993). Thisevidence suggests that TRRE may be a metalloprotease and a serineprotease might act on the activation of TRRE. Most MMPs are actuallysecreted in an inactive soluble proenzyme form (zymogen) which undergoesproteolytic modulation by several serine proteases and an autocatalyticmechanism to be active forms. VanWart and Birkedal-Hansen (1990) Proc.Natl. Acad. Sci. USA 87:5578-5582.

Human monocytes and macrophages have been shown to produce several MMPs;however, no gelatinolytic, caseinolytic, elastinolytic, and type Icollagenolytic activity was detected in crude and partially-purifiedTRRE.

The induction patterns of TRRE and known MMPs by PMA stimulation arequite different. In order to induce MMPs, monocytic U-937 cells,fibrosarcoma HT-1080 cells, or peritoneal exudate macrophages (PEM)usually have to be stimulated for one to three days with LPS or PMA. Onthe other hand, as compared with this prolonged induction, TRRE isreleased very quickly in culture supernatant following 30 min ofPMA-stimulation. As disclosed in Example 2, TRRE is stored in the cellvery close to the cell membrane to be secreted immediately byPMA-stimulation, and TRRE is synthesized very quickly within 2 hoursalso by PMA-stimulation. Therefore, judging from zymography gel data andthe different induction patterns by PMA, TRRE cannot be classified intoone of the pre-existing MMP families, despite their resemblanceregarding metal-requirement and involvement of serine proteases in theiractivation.

Soluble TNF-R has been shown to bind to TNF or LT and form a complexconsisting of 3 sTNF-R with 3 TNF or LT. Banner et al. (1993). Accordingto gel filtration analysis presented above, the profile of TRRE andsoluble p75 TNF-R was quite similar, with both peaks approximately at150 kDa. Since the molecular size of soluble p75 TNF-R was reported tobe 40 kDa, this suggests that sTNF-R exist as a complex formed with TRREor TNF, or otherwise as homo oligomers. The hypothesis that TRRE andsTNF-R form a complex in vitro was confirmed by the experiment that 25%TRRE activity was recovered from soluble p75 TNF-R affinity column. Thismeans that free TRRE has the ability to bind to its catalytic product,sTNF-R. The remaining 75% which did not combine to the affinity columnmay already be bound to sTNF-R or may not have enough affinity to bindto sTNF-R even though it is in a free form.

Although a considerable amount of enzyme product (EP) complex is thoughtto exist in the reacting solution, TRRE retained 86% of its activityafter treated once with excessive substrate, suggesting that thiscomplex can be easily separated when it meets new substrate. This EPcomplex does not seem to inhibit the enzymatic reaction of TRREsignificantly. While sTNF-R is a potent inhibitor against the biologicalactivities of TNF and LT, it was also shown that sTNF-R has another rolein stabilizing TNF activity in vitro. Aderka et al. (1992) J. Exp. Med.175:323-329. Thus sTNF-R might act as a stabilizer not only for TNF, butalso for TRRE by composing complex formation. This EP complex betweenTRRE and sTNF-R may be formed presumably under in vitro conditions,however it is possible that TRRE, sTNF-R and TNF make up several typesof complexes in vivo as well as in vitro, and therefore may havephysiological significance.

EXAMPLE 5 Biological Effect of TRRE

In this Example, the effect and biological significance of TRRE isinvestigated, including (a) substrate specificity and (b) function invitro.

Fluorescein isothiocyanate (FITC)-conjugated anti-CD54, FITC-conjugatedgoat anti-rabbit and mouse antibodies, mouse monoclonal anti-CD30,anti-CD11b and anti-IL-1R (Serotec, Washington D.C.) were utilized inthis study. Rabbit polyclonal anti-p55 and p75 TNF-R were constructedaccording to the method described by Yamamoto et al. (1978) CellImmunol. 38:403-416. THP-1 cells were treated for 30 min with 1,000and/or 5,000 U/ml of TRRE eluted from the DEAE-Sephadex column andtransferred to 12×75 mm polystyrene tubes (Fischer Scientific,Pittsburgh, Pa.) at 1×10⁵ cells/100 μl/tube. The cells were thenpelleted by centrifugation at 350×g for 5 min at 4° C. and staineddirectly with 10 μl FITC-conjugated anti-CD54 (diluted in cold PBS/0.5%sodium aside), indirectly with FITC-conjugated anti-mouse antibody aftertreatment of mouse monoclonal anti-CD11b, IL-1R and CD30 and alsoindirectly with FITC-conjugated anti-rabbit antibody after treatment ofrabbit polyclonal anti-p55 and p75 TNF-R.

THP-1 cells stained with each of the antibodies without treatment ofTRRE were utilized as negative controls. The tubes were incubated for 45min at 4° C., agitated every 15 min, washed twice with PBS/2% FCS,repelleted and then resuspended in 200μg of 1% paraformaldehyde. Theselabeled THP-1 cells were analyzed using a fluorescence activated cellsorter (FACS) (Becton-Dickinson, San Jose, Calif.) with a 15 mW argonlaser with an excitation of 488 nm. Fluorescent signals were gated onthe basis of forward and right angle light scattering to eliminate deadcells and aggregates from analysis. Gated signals (10⁴) were detected at585 BP filter and analyzed using Lysis II software. Values wereexpressed as percentage of positive cells, which was calculated bydividing mean channel fluorescence intensity (MFI) of stained THP-1cells treated with TRRE by the MFI of the cells without TRRE treatment(negative control cells).

In order to test the in vitro TNF cytolytic assay by TRRE treatment theL929 cytolytic assay was performed according to the method described byGatanaga et al. (1990b). Briefly, L929 cells, an adherent murinefibroblast cell line, were plated (70,000 cells/0.1 ml/well in a 96-wellplate) overnight. Monolayered L929 cells were pretreated for 30 min with100, 500 or 2,500 U/ml of partially-purified TRRE and then exposed toserial dilutions of recombinant human TNF for 1 hour. After washing theplate with RPMI-1640 with 10% FCS to remove the TRRE and TNF, the cellswere incubated for 18 hours in RPMI-1640 with 10% FCS containing 1 μg/mlactinomycin D at 37° C. in 5% CO₂. Culture supernatants were thenaspirated and 50 μl of 1% crystal violet solution was added to eachwell. The plates were incubated for 15 min at room temperature. Afterthe plates were washed with tap water and air-dried, the cells stainedwith crystal violet were lysed by 100 μl per well of 100 mM HCl inmethanol. The absorbance at 550 nm was measured using an EAR 400 ATplate reader (SLT-Labinstruments, Salzburg, Austria).

TRRE was originally defined as a protease which truncated the human p75TNF-R that was overexpressed on cDNA-transduced COS-1 cells (C75R). Toinvestigate whether TRRE may truncate not only p75 but also p55 TNF-R onhuman cells, partially-purified TRRE from human THP-1 cells was appliedto THP-1 cells which express low levels of both p55 and p75 TNF-R(approximately 1,500 receptors/cell by Scatchard analysis, data notshown). TRRE eluate from the DEAE-Sephadex column was added to THP-1cells (5×10⁶ cells/ml) at a final TRRE concentration of 1,000 U/ml for30 min. The concentration of soluble p55 and p75 TNF-R in thatsupernatant was measured by soluble p55 and p75 TNF-R ELISA. TRRE wasfound to truncate both human p55 and p75 TNF-R on THP-1 cells andreleased 2,382 and 1,662 pg/ml soluble p55 and p75 TNF-R, respectively(FIG. 15). Therefore, TRRE was capable of truncating human p75 TNF-R onC75R cells and both human p55 and p75 TNF-R on THP-1 cells.

For substrate specificity of TRRE, the cell surface expression of p55and p75 TNF-R, CD54, CD11b, IL-1R, and CD30 on THP-1 cells treated withTRRE was investigated by flow cytometry after labeling THP-1 cells withspecific antibodies as described above. Following treatment of THP-1cells with 5,000 U/ml TRRE, the expression of p55 and p75 TNF-R haddecreased to 8% and 49% (percent control), respectively (FIG. 14), and adose response to TRRE (1,000 and 5,000 U/ml) was seen in the truncationof p55 and p75 TNF-R. No significant change, however, was found in theexpression of CD54, CD11b, IL-1R and CD30 with (5,000 U/ml) and withoutTRRE treatment (FIG. 14). Among the receptors and antigens examined,TRRE was effective against only p55 and p75 TNF-R. The % control wasobtained by dividing mean channel fluoresence intensity (MFI) of stainedTHP-1 cells treated with TRRE by the MFI of the cell without TRRE(control).

In order to investigate the in vitro biological significance of sheddingsTNF-R by TRRE, L929 cytolytic assay, modified by the addition of TRREtreatment, was performed as described above. L929 cells without TRREtreatment were utilized as a negative control. L929 cells werepretreated for 30 min with 100, 500 or 2,500 U/ml TRRE fromDEAE-fractions and then exposed to serial dilutions of recombinant humanTNF for 1 hour. After washing the plate with medium to remove the TRREand TNF, the cells were incubated for 18 hours in 1 μg/ml ActD, stainedwith crystal violet (the live cells stained blue) and then assayed fortheir cytolysis with measuring absorbance at 550 nm. The resultsobtained and depicted in FIG. 15. The ratios of surviving L929 cellspretreated with 0 (negative control), 100, 500 and 2,500 U/ml TRRE with4 ng/ml TNF stimulation to those cells without TNF stimulation were 24%,30%, 38%, and 74%, respectively. TRRE-treated L929 cells were thusprotected against TNF-induced lysis, and more importantly, adose-response for this protection was detected.

The substrate-specificity of TRRE was investigated using membranereceptors and antigens other than the two TNF-Rs. These receptors andantigens are expressed at sufficient levels on THP-1 cells to bedetected by FACS analysis including (i) IL-1R, whose soluble form isknown to be produced by proteolytic cleavage, (ii) CD30 (ki-1), whichbelongs to the same receptor family as TNF-R (TNF-R/NGF-R superfamily)and whose soluble form is produced presumally by a Zn²⁺-dependentmetalloprotease, (iii) CD54 (ICAM1), which belongs to immunoglobulinsuperfamily of adhesion molecules including VCAM-1 and is known to havea soluble form, and (iv) CD11b, which belongs to the integrin family ofadhesion molecules and which has not been shown to have a soluble form.The FACS analysis presented above revealed that TRRE is very specific toonly the cleavage of both TNF-Rs and did not affect any other membranereceptors and antigens which have soluble forms. In addition, theability of TRRE to cleave both TNF-Rs was supported by the soluble p55and p75 TNF-R ELISA data presented herein.

TRRE down-regulated the expression of TNF-R on the cell-surface of L929cells in the TNF binding assay. Pretreatment with TRRE protected L929cells from the killing activity of TNF. Thus, TRRE may control TNFactivity by two methods; by reducing the number of TNF-R or by producingsTNF-R which bind and inactivate TNF. Cleavage of TNF-R by TRRE maypossibly protect TNF-sensitive cells and organs in diseases associatedwith high levels of TNF. On the other hand, while high serum levels ofTNF and sTNF-R are often associated with various types of cancers,cleavage of TNF-R by TRRE may condition cancer cells more resistant tothe effects of TNF.

EXAMPLE 6 Purification of TRRE

TRRE was purified to apparent homogeneity by 100% saturated ammoniumsulfate precipitation, DEAE-Sephadex chromatography, and native PAGE.Partially-purified TRRE was fractionated by SDS-PAGE and several proteinbands were apparent. Two protein bands, at 60 kDa and 37 kDa, wereselected as possible TRRE candidates.

THP-1 cells were cultured in 8 roller bottles (Corning, Corning, N.Y.)per preparation with 500 ml of 10% FCS-contained RPMI-1640 per rollerbottle. The cells were collected for the induction of TRRE when the celldensity reached approximately 1×10⁶ to 1.5×10⁶ cells/ml. The cells werethen incubated in 1% FCS-contained medium with 10⁶ M phorbol12-myristate 13-acetate PMA (Sigma) for 30 min and were washed to obtainPMA-free sample. After washing serum free medium, the cells wereincubated for an additional 2 hours in a 4 liter, 1% FCS containingmedium with PMA and the supernatants were collected as the source of theenzyme.

The cell-free supernatants were concentrated by the 100% saturatedammonium sulfate precipitation method. All of the following procedureswere performed at 4° C. 69.7 g of solid ammonium sulfate per 100 ml wasslowly added to the collected supernatant over 4 hours with gentlestirring followed by an additional 1 hour of stirring. The precipitatewas collected by centrifugation at 10,000×g for 30 min and redissolvedin PBS at approximately two times the volume of the pellet. There-dissolved precipitates was then dialyzed against a buffer of 10 mMTris-HCl, 60 mM NaCl, pH 7.0 (buffer 1) with dialysis tubing which had anominal molecular weight cut-off (NMWC) of about 6,000 to 8,000 for 60hours.

The concentrated samples were diluted in the same volume of a buffer of50 mM Tris-HCl, 60 mM NaCl, pH 8.0 (buffer 2) and then applied onto ananion-exchange chromatography, DEAE-Sephadex A-25 (Pharmacia Biotech,Uppsala, Sweden) (2.5×10 cm), which was previously equilibrated withbuffer 2. Following washing of the column with (150 ml) of buffer 2, thesample was eluted with an ionic strength linear gradient (total volume;250 ml) of 60 mM (buffer 2) to 250 mM NaCl, 50 mM Tris-HCl, pH 8.0. Theflow rate was 1 ml/min, and 4-ml fractions of sample A and 3-mlfractions of sample B were collected. Each fraction was measured forabsorbance at 280 nm and assayed for TRRE activity. Several DEAEfractions with the highest specific TRRE activity (the highest value ofTRRE units/A280) were collected and applied to the further steps.

These active DEAE fractions were centrifugally concentrated toapproximately 500 μl with Centriprep-10 filter (10,000 MW cut-offmembrane) (Amicon). This concentrated sample was then applied to severallanes of a 6% polyacrylamide gel electrophoresis (PAGE) (15×10 cm) undernon-denaturing native conditions with cooling to recover the biologicalactivity of TRRE after electrophoretic separation. One complete lane wascut off vertically from the side of the native PAGE slab gel and stainedwith a silver staining kit (Bio-Rad). The remainder of the native PAGEgel was then sliced horizontally into 5 mm strips and each strip waseluted in 1 ml of PBS at 4° C. overnight with shaking. Each eluate wasassayed for TRRE activity. The TRRE activity of each eluate was comparedwith protein bands of the silver-stained native gel for the localizationof TRRE. Next, each TRRE active fraction eluted from native PAGE gel wascentrifugally concentrated to approximately 50 μl with Microcon-10filter (10,000 MW cut-off membrane) (Amicon). These concentrated sampleswere applied to 8% SDS-PAGE under denaturing conditions, and stainedwith Coomassie Brilliaht Blue R-250 (CBB). The stained gel was then usedto determine the existence of a correlation between TRRE activity andthe intensity of the protein bands.

With 4 L of the original THP-1 cell supernatant (8 roller bottles),representing one lot, the PMA-stimulated THP-1 cells were prepared at adensity of 1.5×10⁶ cells/ml, and 3,679±144 ml and 3,623±118 ml of theTRRE sources were obtained for sample A and sample B, respectively(mean±standard error). After 100% saturated ammonium sulfateprecipitation, sample A and sample B were dissolved in 49±5 ml and4.5±1.1 ml of PBS and reached a final volume of 109±12 ml after 60 hoursof dialysis and 13.2±2.7 ml after 24 hours of dialysis, respectively.The final fold-concentrations of sample A and sample B were 34.2±2.3times and 268±26 times, respectively. Sample A had 4,327±1,150 U/ml ofTRRE and a total amount of (15.9±2.5)×10⁶ U in the original supernatant.After dialysis for 60 hours, the concentrated sample A had(12.1±2.9)×10⁴ U/ml and a total of (13.2±2.1)×10⁶ U of TRRE. Therefore,81.6±8.1% TRRE was recovered in sample A through 100% saturated ammoniumsulfate precipitation. For sample B, TRRE activity in the originalsupernatant was unable to be assayed because of the contamination of10⁻⁶ M PMA. After ammonium sulfate precipitation followed by intensivedialysis for 24 hours to remove ammonium sulfate and PMA, theconcentrated sample B had (21.9±2.1)×10⁴ U/ml and a total of(28.9±3.8)×10⁵ U of TRRE which corresponded to about one fifth of totalTRRE of sample A.

One lot of dialyzed sample A was loaded onto a DEAE-Sephadex column with4 aliquots. With this column, both proteins and TRRE activity eluted assimilarly-shaped single broad peaks which tapered gradually in thelatter fractions. The peak protein fraction (A280) and TRRE activityfraction was always found near Fr. 20 and Fr. 15, respectively (FIG.16). Thus, TRRE was eluted in 4 or 5 fractions prior to the mainproteins which is predominantly comprised of bovine serum albumin (BSA).The concentration of the highest TRRE fraction was (25.5±2.4)±10³ U/ml.With this column, 0.3±0.2%, 4.5±1.1%, and 35.1±6.3% TRRE activity wererecovered in flow through, washing, and all fractions, respectively,compared to the original TRRE activity loaded on the column (100%).Among the total proteins recovered (100%), 21.8±2.5%, 32.6±2.8%, and45.6±5.3% proteins were obtained in flow through, washing, and allfractions, respectively.

On the other hand, due to extremely low amounts of proteins, two lots ofsample B were combined and loaded onto a DEAE-Sephadex column per columnpurification. TRRE activity eluted as a broad single peak as in sample A(data not shown). The elution of proteins, however, held at a low valueonce it reached a certain point, and so no significant peak wasdetected. The peak of TRRE activity was at Fr. 18 and the proteinsgradually reached their highest value near Fr. 40. With this column,0±0%, 2.2±0.4%, and 11.2±1.3% TRRE activity were recovered in flowthrough, washing, and all fractions, respectively, compared to theoriginal TRRE activity loaded on the column (100%). 9.5±3.7%, 42.4±5.5%,and 48.1±6.1% of the total proteins recovered (100%) were located in theflow through, washing, and all fractions, respectively, very similar tothe percentages of sample A. The recovered efficacy of TRRE in sample B,however, was lower than sample A.

Several DEAE fractions with the highest relative TRRE activity wereconcentrated and loaded onto a native PAGE. TRRE activity of sample Awas detected in 4 fractions (strips) from Fr. 8 to 11 in the nativePAGE. The highest TRRE activity was at Fr. 9 or 10 which had(9.3±1.6)×10³ U/ml activity and the total recovery of TRRE activity was16.2±4.1% through concentration and native PAGE. The highest TRREfraction was found with a tight group of several bands detected bysilver staining of the native PAGE gel. On the other hand, TRRE activityof sample B was detected in 3 fractions of Fr. 13 to 15 in native PAGE.The total recovery rate of TRRE activity from these 3 fractions was8.7±1.0% through concentration and native PAGE. Only 2 or 3silver-stained protein bands were detected at Fr. 13-15 of theTRRE-active eluates in the native PAGE.

SDS-PAGE of concentrated active eluates from the native PAGE revealedseveral protein bands in sample B. The TRRE eluate with highest-activityhad protein bands at approximate 120 kDa, 60 kDa and 37 kDa, whileseveral other TRRE-active eluates had protein bands at 70 kDa, 55 kDa,40 kDa and 20 kDa including the 120 kDa, 60 kDa and 37 kDa bands. Morebands were detected in sample A due to the contaminated proteins fromFCS. The intensity of the protein bands of TRRE should correlate withTRRE activity. Thus, the 60 kDa and 37 kDa bands were the strongestcandidates as TRRE because of their corresponding increase in intensitywith higher levels of TRRE. The 37 kDa band appeared to correlate betterwith TRRE than the 60 kDa band.

Two types of enzyme sources of TRRE were prepared for its purification.First, in sample A, TRRE was induced to high levels from PMA-stimulatedTHP-1 cells in 1% FCS-contained medium without PMA. This was a goodsource of TRRE, but high protein contamination from FCS was observed. Inthe second source, named sample B, TRRE was induced much less thansample A from THP-1 cells in PMA-contained medium without FCS. Here avery pure protein sample was obtained without FCS-contamination but muchless TRRE activity was detected. Moreover, the recovery of TRRE activityin sample B was markedly less than sample A at every step of thepurification procedure including dialysis, centrifugal concentration bya membrane-filter, DEAE column and native PAGE. Therefore, aninsufficient amount of purified TRRE was isolated for AA sequencing withthe procedure of sample B. However, sample B was helpful in identifyingthe possible TRRE band at the final step of SDS-PAGE due to its purity.Thus, after a potential band in the SDS-PAGE was identified from themore pure sample B, sample A was utilized to prepare enough of purifiedTRRE for AA sequencing.

In DEAE-Sephadex column chromatography TRRE eluted in earlier fractionsthan the main proteins, most of which considered as BSA. SinceDEAE-Sephadex also has a gel-filtration effect in addition to ananion-exchange effect, the molecular size of TRRE may be larger than BSAwhose molecular size is about 69 kDa. However, the finding that themolecular sizes of possible TRRE candidates at the final purificationstep were 37 kDa and 60 kDa may also support data presented hereinindicating that TRRE exists as a complex formed with sTNF-R and TNF, asTRRE displayed the ability to combine with sTNF-R in Chapter III.Another possibility is that TRRE may exist as a homo or hetero oligomerpresumably consisting of 37 kDa and/or 60 kDa monomer.

In this Example, TRRE was shown to easily lose its activity throughevery purification step especially at low protein and saltconcentrations. We have performed several other purification proceduresin addition to those described in this Example. For example, TRRE samplewas subjected to C4 reverse-phase high-performance liquid chromatography(HPLC) in the 5 to 95% gradient of acetonitrile with moderateresolution. TRRE activity, however, was completely inactivated inacetonitrile, although a small amount of the activity was restored afterlyophilization. Fast protein liquid chromatography (FPLC) was thenapplied and turned out to be inappropriate for separating TRRE frommajor proteins or for large-scale purification. To purify TRREspecifically, we tried two kinds of affinity chromatography such assoluble p75 TNF-R affinity column mentioned in Chapter III andanti-soluble p75 TNF-R antibody affinity column by taking advantage ofthe ability of TRRE to make a complex form with sTNF-R. These methodswere acceptable for obtaining pure TRRE but were unsuitable for handlinglarge amounts necessary for AA sequencing. Taking all these factors intoaccount, our present purification scheme has worked quite efficiently torecover high specific TRRE activity and was capable of handling bothlarge amounts and volume of protein samples necessary for AA sequencing.

EXAMPLE 7 Purification of TRRE

The following protocol was used in purification of TRRE.

-   1. Supernatant from large scale cell culture of 10-6M PMA stimulated    THP-1-   ↓-   2. Ammonium sulfate precipitation-   ↓-   3. DEAE-Sephadex chromatography-   ↓-   4. 6% Native PAGE-   ↓-   5. 10% SDS-PAGE-   ↓-   6. Transfer to nitrocellulose membrane-   ↓-   7. Trypsin digestion-   ↓-   8. Reverse-phase HPLC C18 column-   ↓-   9. Analysis of amino acid sequence

Step 1: Large scale cell culture. THP-1 were cultured in 4.0 L serumcontaining medium until the cell density reached 1×10⁶ to 1.5×10⁶cell/ml. The cells were incubated in 1% FCS containing medium with 10⁻⁶MPMA (phorbol 12-myristate 13-acetate) (Sigma Chemical, St. Louis, Mo.)for 30 min. After washing in serum free medium, the cells were incubatedfor additional 2 hours in 4 L 1% FCS containing medium without PMA andthe supernatant was collected as the source of the enzyme.

Step 2: Ammonium sulfate precipitation. The supernatant was concentratedby the 100% saturated ammonium sulfate precipitation method described inExample 4. The precipitate was collected and redissolved in PBS anddialyzed against 10 mM Tris-HCl, 60 mM NaCl (pH 7.0) for 60 hours.

Step 3: DEAE-Sephadex Chromatography. The concentrated samples werediluted in the same volume of 50 mM Tris-HCl, 60 mM NaCl (pH 8.0) andapplied onto an anion-exchange chromatography, DEAE-Sephadex A-25(Pharmacia Biotech, Uppsala, Sweden) column. The samples were elutedwith a sodium linear gradient (60 mM to 250 mM NaCl). Several fractionswith the highest specific TRRE activity were collected.

Step 4: Native PAGE. The active DEAE fractions were centrifugallyconcentrated to 500 μl with a Centriprep-10 filter (10,000 MW cut-offmembrane) (Amicon). This concentrated sample was applied to 6% PAGEunder non-denaturing native conditions. The gel was sliced horizontallyinto 5 mm strips and each was eluted into 1 ml PBS. Each TRRE activefraction was centrifugally concentrated to 50 μl with a Centriprep-10filter.

Step 5: SDS-PAGE and Protein blotting. The concentrated active sampleseluted from native PAGE were loaded on 10% SDS-PAGE. The proteins werethen electrophoretically transferred to nitrocellulose membrane andstained with 0.1% Ponceau S.

Step 6: Preparation of peptide fragments for microsequencing. Each bandwas cut out from the nitrocellulose membrane and digested with 1 μgtrypsin (Boehringer) in digestion buffer (0.1 M Tris-HCl pH 8.0, 1 mMCaCl₂, 10% (v/v) acetonitorile) overnight.

The digested samples were spun down and the supernatants were injectedonto reverse phase HPLC C₁₈ column (4.6×250 mm). The peptide fragmentswere eluted with a linear gradient of 0-60% acetonitrile containing 0.1%trifluoroacetic acid. Several peak fractions were collected andsubjected to amino acid sequence analysis using an Applied Biosystems,Inc. peptide sequencer.

Candidates of the TRRE

SDS-PAGE of concentrated active eluates from the native PAGE revealedseveral protein bands. The TRRE eluate with highest activity had proteinbands at approximate 120 kDa, 60 kDa, and 37 kDa. The 60 kDa and 37 kDabands were the strongest candidates as TRRE because of theircorresponding increase in intensity with higher levels of TRRE activity.Thus, at first the 60 kDa band (p60) of blotting membrane stained with0.1% Ponceau S were cut out and subjected to further analysis.

EXAMPLE 7 Use of TRRE in Treating Septic Shock

The following protocol was followed to test the effects of TRRE inpreventing mortality in test animals which were treated withlipopolysaccharides (LPS) to induce sepsis and septic shock.

Generally, mice were injected with lethal or sublethal levels of LPS,and then with a control buffer or TRRE. Samples of peripheral blood werethen collected at intervals to establish if TRRE blocked TNF-inducedproduction of other cytokines in the bloodstream. Animals were assessedgrossly for the ability of TRRE to block the clinical effects of shockand then euthanized and tissues examined by histopathological methods.

More specifically, adult Balb/c mice, the traditional animal model forseptic shock studies [see, for example, Mack et al. (1997) J. Surg. Res.69:399-407; and Seljelid et al. (1997) Scand. J. Immunol. 45:683-7],were placed in a restraining device and injected intravenously via thetail vein with a 0.1 ml solution containing 10 ng to 10 mg of LPS inphosphate buffer saline (PBS). These levels of LPS induce mild to lethallevels of shock in this strain of mice. Shock results from changes invascular permeability, fluid loss, and dehydration, and is oftenaccompanied by symptoms including lethargy, a hunched, stationaryposition, rumpled fur, cessation of eating, cyanosis, and, in seriouscases, death within 12 to 24 hours. Control mice received an injectionof PBS. Different amounts (2,000 or 4,000 U) of purified human TRRE wereinjected IV in a 0.1 ml volume within an hour prior to or after LPSinjection. Serum (0.1 ml) was collected with a 27 gauge needle and 1 mlsyringe IV from the tail vein at 30, 60 and 90 minutes after LPSinjection. This serum was heparinized and stored frozen at −20° C.Samples from multiple experiments were tested by ELISA for the presenceof sTNF-R, TNF, IL-8 and IL-6. Animals were monitored over the next 12hours for the clinical effects of shock. Selected animals wereeuthanized at periods from 3 to 12 hours after treatment, autopsied andvarious organs and tissues fixed in formalin, imbedded in paraffin,sectioned and stained by hematoxalin-eosin (H and E). Tissue sectionswere subjected to histopathologic and immunopathologic examination.

As shown in FIG. 19, mice injected with LPS alone or LPS and a controlbuffer demonstrated rapid mortality. 50% of the test animals were deadafter 8 hours (LPS) or 9 hours (LPS plus control buffer), and 100% ofthe animals were dead at 15 hours. In contrast, when injections of LPSwere accompanied by injections of a 2,000 U of TRRE, death was delayedand death rates were lower. Only 40% of the animals were dead at 24hours. When 4,000 U of TRRE was injected along with LPS, all of theanimals had survived at 24 hours. Thus, TRRE is able to counteract themortality induced by LPS in test animals.

EXAMPLE 8 Effect of TRRE on the Necrotizing Activity of Human TNF InVivo

The following protocol was followed to test the effects of TRRE inaffecting tumor necrosis in test animals in which tumors were produced,and in which TNF was subsequently injected.

Generally, on Day 0, cutaneous Meth A tumors were produced on theabdominal wall of fifteen BALB/c mice by intradermal injection of 2×20⁵Meth A tumor cells.

On Day 7, the mice were divided into three groups of five mice each andtreated as follows:

-   -   Group 1: Injected intravenously with TNF (1 μg/mouse).    -   Group 2: Injected intravenously with TNF (1 μg/mouse) and        injected intratumorally with TRRE (400 units/mouse, 6, 12 hours        after TNF injection).    -   Group 3: Injected intravenously with TNF (1 μg/mouse) and        injected intratumorally with control medium (6, 12 hours after        TNF injection).

On Day 8, tumor necrosis was measured with the following results:

% of necrosis Group 1: 100 (5/5) Group 2:  20 (1/5) Group 3:  80 (4/5)

Therefore, injections of TRRE greatly reduced the ability of TNF toinduce necrosis in Meth A tumors in BALB/c mice.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications can be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention which is delineated by the appended claims.

1. A method for enriching TNF receptor releasing (TRRE) activity,comprising: a) obtaining cells that express p55 or p75 TNF receptor(TNF-R) on the cell surface; b) separating a cell supernatant containingTRRE activity into different fractions according to physical or chemicalcharacteristics; c) contacting the cells from step a) with the fractionsfrom step b); d) determining which of the separated fraction(s) causerelease of TNF-R from the cells; and e) collecting the fraction(s)determined to cause release of TNR-R in step d); whereby TRRE activityis enriched.
 2. A method for characterizing a fraction enriched for TRREactivity from a cell supernatant, comprising obtaining fraction(s)enriched for TRRE activity according to the method of claim 1, and thendetermining amino acid sequence(s) contained in said enrichedfraction(s).
 3. The method of claim 1, wherein the TNF receptorexpressing cells obtained in step a) have been transfected orrecombinantly transformed to express the p75 TNF receptor.
 4. The methodof claim 1, wherein the TNF receptor expressing cells obtained in stepa) have been transfected or recombinantly transformed to express the p55TNF receptor.
 5. The method of claim 1, wherein the supernatant wasobtained from cells selected from the cell lines THP-1, U-937, HL-60,ME-180, MRC-5, Raji, and K-562; and normal human monocytes.
 6. Themethod of claim 5, wherein the supernatant was obtained from theselected cells by simulating the cells with phorbol myristate acetate(PMA), IL-10, or epinephrine.
 7. The method of claim 1, wherein theseparating of the TRRE activity in step b) comprises fractionating thesolution by a combination of ion exchange chromatography and separationby molecular weight.
 8. The method of claim 1, wherein said determiningin step d) comprises measuring binding capacity for TNF on the cellsurface.
 9. The method of claim 1, wherein said determining in step d)comprises measuring the concentration of soluble TNF-R released from thecell surface.
 10. The method of claim 1 wherein the method furthercomprises concentrating the fraction(s) collected in step d).